Methods of providing modulators of tau aggregation

ABSTRACT

The present invention relates generally to methods for selecting or designing a compound for modulating the aggregation of a Tau protein. The method comprising using computer-implemented molecular modelling means to compare the three-dimensional structure of a candidate compound with a three-dimensional structure of at least a part of the Tau protein comprising amino acids 315-378 and determine whether the candidate compound is able to simultaneously form non-covalent interactions with two or more of Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350, Ser352, Ile354, Lys369, Ile371, Glu372, Phe378 and Thr373. A candidate compound that is able to form said interactions is predicted to modulate the aggregation of the Tau protein or truncated form thereof. Methods using a three-dimensional structural model of at least a part of the Tau protein comprising amino acids 315-378, wherein the model is an intermediate in the aggregation process of the part of the Tau protein with a paired helical filament (PHF) are also described, as are computing systems and products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a National Stage filing under 35 U.S.C. § 371 of International PCT Application No. PCT/EP2021/068718, filed Jul. 6, 2021, which claims priority to Great Britain Application No. 2010679.5, filed Jul. 10, 2020. The contents of these applications are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates generally to methods of using the structure coordinates of a Tau binding pocket. In particular, the invention provides a three-dimensional model of a Tau binding pocket and means for identifying, selecting and/or designing modulators of Tau aggregation by rational drug design. The invention also relates generally to using the structure coordinates of a Tau protein that is an intermediate in the process of folding of the Tau protein from a compact conformation comprising the binding pocket to an aggregated confirmation in a paired helical filament.

BACKGROUND ART

Disorders related to tau, collectively referred to as neurodegenerative tauopathies (Lee et al., 2001) represent a group of diseases of protein aggregation (Buee et al., 2000). Alzheimer's disease (AD) is part of this group of neurodegenerative diseases. Conditions of dementia such as Alzheimer's disease (AD) are frequently characterised by a progressive accumulation of intracellular and/or extracellular deposits of proteinaceous structures such as β-amyloid plaques and neurofibrillary tangles (NFTs) composed of tau, in the brains of affected patients. The appearance of these lesions largely correlates with pathological neuronal degeneration and brain atrophy, as well as with cognitive impairment (see, e.g., Mukaetova-Ladinska et al., 2000). In AD, tau protein self-assembles to form paired helical filaments (PHFs) and straight filaments that constitute the neurofibrillary tangles within neurons and dystrophic neurites in brain. Protein misfolding to form amyloid fibrils is a hallmark of many different diseases collectively known as the amyloidoses, each of which is characterised by a specific precursor protein (Sipe, 1992).

Tau exists in alternatively-spliced isoforms, which contain three or four copies of a repeat sequence corresponding to the microtubule-binding domain (see, e.g., Goedert, M., et al., 1989). The tau species isolated from proteolytically stable PHF-core preparations from AD brain tissue comprise a mixture of fragments derived from both three- and four-repeat isoforms, but restricted to the equivalent of three repeats, with an N-terminus at residues Ile-297 or His-299 and the C-terminus at residue Glu-391, or at homologous positions in other species (Wischik et al., 1988; Jakes et al., 1991). The present inventors have previously shown that the fragment 297-391 (referred to as dGAE) self-assembles to form Paired helical filament-like structures under specific conditions (Al-Hilaly et al., 2017) and that assembly is enhanced under reducing conditions (in the presence of DTT) or at high concentrations. Assembly is accompanied by a conformational change from random coil in solution to beta sheet structure in the insoluble fraction (Al-Hilaly et al., 2017). Residues 306-378 from this same molecule (referred to as “dGAE73” herein) have been visualised in electron density and confirmed as forming part of the core of the PHF using cryo-electron microscopy analysis of PHFs extracted from AD brain tissue (Fitzpatrick et al., 2017).

However, despite the availability of structural data on amyloid fibrils formed by different proteins and from PHFs, the elucidation of a mechanism of amyloid fibril formation from disjoint monomers remains elusive (Sipe 1992; Chiti & Dobson 2006; Roberts 2007; Kelly 2000). To date there has been no agreed description of the primary stages of nucleation from a pool of disjoint soluble oligomeric species (Roberts 2007, Kodali & Wetzel, 2007; Serio et al., 2000; Modler et al., 2004; Glabe 2008; Meisl et al., 2016). A number of amyloidogenic precursors and oligomeric states have been identified (Campioni et al., 2010; Novo et al., 2018; Kayed et al., 2003; Buccianti et al., 2002 and 2004; Gerson et al., 2016), yet isolation and further characterisation of these structurally heterogeneous and transient aggregates remains a challenge. Evidence of soluble protein oligomers as the primary cause of cell impairment and dysfunction in the pathogenesis of tauopathies has been supported through biochemical and biophysical experiments and in vivo assays (Macdonald et al., 2019; Lasagna-Reeves et al., 2012; Gerson et al., 2016).

The present inventors have shown previously that the methylthioninium (MT) species, which can exist in oxidized (MTC, FIG. 12A) and reduced leuco-MT (LMT; FIG. 12C) forms, is able to act as a tau aggregation inhibitor in cell-free, cell based and tau transgenic mouse models of tau aggregation (Harrington et al., 2015; Wischik et al., 1996; Melis et al., 2015; AI-Hilaly et al., 2018). Recent findings indicate that LMT is the active moiety required for inhibition of aggregation of the core tau unit of the PHF (AI-Hilaly et al., 2018). This finding supports the clinical evidence that the stable reduced salt form of the MT moiety (hydromethylthioninium mesylate, LMTM, FIG. 12B) appears to be effective at a dose 20-fold lower than the minimum effective dose previously identified using the oxidized MT+ form (Wischik et al., 2015; Schelter et al., 2019).

As tau aggregation pathology and cognitive decline are closely associated (Okamura et al., 2014; Chien et al., 2013; Mukaetova-Ladinska et al., 2000; Grober et al., 1999; Wilcock and Esiri, 1982; Duyckaerts et al., 1997; Bancher et al., 1993 & 1996; Arriagada et al., 1992), treatment based on the use of inhibitors of tau aggregation offers a potentially promising therapeutic approach for AD (Wischik et al., 2018). A fundamental component for the development of new chemical entities for the treatment of neurodegenerative tauopathies is an understanding of the structure of key intermediates in this aggregation process, and how compounds such as LMT can interfere with this process. This would enable the development of a structure-based hypothesis for ligand design.

DISCLOSURE OF THE INVENTION

In an attempt to address the above-mentioned needs, the present inventors devised multistep computational protocols to explore the conformational flexibility of a previously reported truncated tau fragment corresponding to one of the species isolated from proteolytically stable PHF preparations (residues ²⁹⁷Ile-Glu³⁹¹) and referred to as “dGAE” (Wischik et al., 1988; Novak et al., 1991; Novak et al., 1993). dGAE assembles spontaneously in physiological conditions in vitro into filaments that closely resemble native PHFs and straight filaments from AD brain morphologically (AI-Hilaly et al., 2017; AI-Hilaly et al., 2020). The analyses were aimed at understanding the flexibility of dGAE, its aggregation into PHFs and the potential of small molecules to inhibit PHF assembly and to cause PHF disaggregation in vivo. In an effort to understand the mechanism of how small molecules could inhibit dGAE assembly into fibrils, the conformation space of a single Tau97 monomer (comprising dGAE and preceding residues ²⁹⁵Asp²⁹⁶Asn) was evaluated with atomistic molecular simulation. The inventors hypothesized that LMT could complex to dGAE monomers and prevent fibril formation. To test this hypothesis, representative protein structures were sampled from the conformational ensemble of apo Tau97. From this, the inventors were able to identify a unique cryptic pocket which would bind to LMT and form a stabilised complex. From the conformation of the complex a pharmacophore model was built and a series of de novo designed compounds were synthesised in order to assess whether stabilising dGAE oligomers would result in inhibition of Tau aggregation. Compounds were assessed in a cell-based tau aggregation assay. More than half of the assayed molecules showed sub micro molar activity. From this, the inventors were able to rationalise the structure-activity relationship from this series of Tau assembly inhibitors.

Thus, the present invention is based, in part, on the discovery of a ligand binding pocket in the Tau protein structure, wherein binding of a ligand in the pocket stabilises the Tau protein in a conformation that is not prone to aggregation into paired helical filaments (PHFs). Interactions between the ligand and any of residues Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350, Ser352, Ile354, Lys369, Ile371, Glu372, Phe378 and Thr373 of Tau were found to be particularly important in stabilising the ligand bound conformation.

The formation of PHF was then analysed through immunostaining dot blots and ELISA assays. These showed that in vitro assembly of PHFs is associated with loss of immunoreactivity with antibodies directed against the PHF core. Furthermore, loss of immunoreactivity could be prevented by including LMT during assembly. Starting from the stabilised conformation of dGAE73 (residues 306-378), a molecular simulation was performed to examine assembly onto a preformed PHF. This simulation supported findings from the dot blots for the assembly of PHFs from stabilized dGAE oligomers. The simulation identified 3 key stages in assembly of PHFs. The first is the electrostatic attraction of oligomers to PHFs and the anchoring of a hairpin turn. This is followed by a proline trans-cis-trans switch which allows for the final step of the formation of stabilising cross-β sheets through hydrophobic zippering of the N- and C-terminal tails of the protein structure. Taken together, the structure-activity results and the dot blots provide strong evidence for the binding of small molecules to the cryptic pocket proposed in our dGAE stabilised conformation, which finds uses in a structure-based approach to design further inhibitors of tau assembly.

Thus the present invention is based, in other aspects, on the discovery of a folding process through which the Tau protein in the compact folded state that includes the binding pocket aggregates with a paired helical filament, where binding of a ligand that inhibits the formation of intermediates in the folding process may stabilise the Tau protein in a conformation that is not prone to aggregation into PHFs. A series of conformational changes from a compact folded state to an aggregated state were found to be particularly important. These conformational changes are such that:

-   -   (i) residues Val337-Gln355 form a hairpin loop that moves to         align with alternating positively charged and negatively charged         sidechain stacks in the hairpin loop of the PHF;     -   (ii) residue Pro332 switches between a trans and a cis         configuration;     -   (iii) residues 355-378 and 306-318 move to form stabilising         cross-β sheets with corresponding residues of the PHF through         hydrophobic zippering.

In embodiments, residue Pro332 switching between a trans and a cis configuration causes residues His329, His330 and Lys331 to move close enough to PHF to establish interactions with the partner residues in the next layer of the PHF stack through hydrophobic stacking and a strong hydrogen bond between the side chains of His330 in a preformed layer of the PHF stack and Thr361 of the newly formed dGAE layer on the PHF stack.

In embodiments, step (iii) comprises the following steps: (iii)(1) residues 355-360 move to establish a zipper with the corresponding anti-parallel residues of the PHF; (iii)(2) residues 361-367 move to establish a zipper with the corresponding anti-parallel residues of the PHF; and (iii)(3) residues 306-318 and 368-378 move to form a cross-beta sheet conformation with corresponding residues of the PHF.

According to a first aspect, there is provided a method for selecting or designing a compound for modulating the aggregation of a Tau protein or a truncated form thereof, the method comprising using computer-implemented molecular modelling means to:

-   -   compare the three-dimensional structure of a candidate compound         with a three-dimensional structure of at least a part of the Tau         protein comprising amino acids 315-378 of SEQ ID NO:1 or a         variant or derivative thereof that is structurally equivalent to         said part; and     -   determine whether the candidate compound is able to         simultaneously form non-covalent interactions with two or more         of Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350,         Ser352, Ile354, Lys369, Ile371, Glu372, Phe378 and Thr373, of         SEQ ID NO:1, or equivalent amino acids in a variant or         derivative, wherein a candidate compound that is able to form         said interactions is predicted to modulate the aggregation of         the Tau protein or truncated form thereof.

According to a related aspect, there is provided a method for selecting or designing a compound for modulating the aggregation of a Tau protein or a truncated form thereof, the method comprising using computer-implemented molecular modelling means to:

-   -   compare the three-dimensional structure of a candidate compound         with a three-dimensional structure of at least a part of the Tau         protein comprising amino acids 315-378 of SEQ ID NO:1 as defined         in Table 1, or a variant or derivative thereof that is         structurally equivalent to said part; and     -   determine whether the candidate compound is able to form         non-covalent interactions with the part of the Tau protein         having the structure defined in Table 1 which stabilise said         structure, wherein a candidate compound that is able to form         said interactions is predicted to modulate the aggregation of         the Tau protein or truncated form thereof.

As used herein, the step of comparing the three-dimensional structure of a candidate compound with a three-dimensional structure of at least a part of the Tau protein comprises providing the three-dimensional structure of the candidate compound, providing the three-dimensional structure of the part of the Tau protein, and obtaining a molecular model that includes both three-dimensional structures.

In embodiments, the non-covalent interactions comprise interactions with one or more, optionally with two or more of: Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350, Ser352, Lys369, Ile371, Glu372, and Thr373, of SEQ ID NO:1, or equivalent amino acids in a variant or derivative. Non-covalent interactions with the above-mentioned residues of the Tau protein were found to stabilise a compact conformation of the Tau protein (an example of which is provided by the coordinates defined in Table 1), reducing the propensity of the Tau protein to aggregate.

In embodiments, the non-covalent interactions comprise at least a non-covalent interaction with Lys347. Indeed, a potent inhibitor of Tau aggregation, compound 16, was identified by the inventors as binding the Tau protein by establishing an interaction with Lys347, thereby stabilising a conformation of the protein that has reduced propensity to aggregate. Further potent inhibitors of Tau aggregation were also found to interact with Lys347, including LMT, compound 17, compound 12, compound 1, compound 7, compound 5, compound 14, compound 2, compound 8, compound 13 and compound 18.

In embodiments, the non-covalent interactions comprise at least a non-covalent interaction with Lys343. Indeed, a potent inhibitor of Tau aggregation, compound 11, was identified by the inventors as binding the Tau protein by establishing an interaction with Lys343, thereby stabilising a conformation of the protein that has reduced propensity to aggregate. Further potent inhibitors of Tau aggregation were also found to interact with Lys343, including LMT, compound 17, compound 12, compound 3, compound 14, compound 10, compound 13 and compound 18.

In embodiments, the non-covalent interactions comprise at least a non-covalent interaction with Thr373. Indeed, a potent inhibitor of Tau aggregation, compound 9, was identified by the inventors as binding the Tau protein by establishing an interaction with Thr373, thereby stabilising a conformation of the protein that has reduced propensity to aggregate. Further potent inhibitors of Tau aggregation were also found to interact with Thr373, including LMT, compound 3, compound 4, and compound 15.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more of Lys343, Leu315, Lys347, Glu372, and Thr373, of SEQ ID NO:1.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with one or more of Leu315, Val350, and Ile371 of SEQ ID NO:1. Interactions with these residues are preferably hydrophobic interactions, involving the side chain of the residue. Indeed, multiple potent inhibitors of Tau aggregations, such as compounds 16, 9 and 11, were identified by the inventors as binding the Tau protein by establishing an interaction with these residues.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Lys347, Lys343, Ser352 and Thr373 of SEQ ID NO: 1. A known inhibitor of Tau aggregation, LMT was identified by the inventors as binding the Tau protein in a cryptic binding pocket, where it establishes interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Lys347, Lys343, Ser341, Leu315, Ile371, Glu372 and Phe346 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 17, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Lys347, Lys343, Ile371, and Glu372 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 12, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Lys347, Ile371, Glu342, Phe346 and Glu372 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 1, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Leu315, Lys347, Ile371, Glu342, Phe346 and Glu372 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 7, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with at least Lys343 and Thr373 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 3, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Glu372, Lys369, and Thr373 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 7, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Lys343, Lys347, Ile371, Glu342, Phe346 and Glu372 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 5, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Leu315, Lys347, and Lys343 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 14, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Lys343, Lys347, and Glu372 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 2, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Lys347, Glu342, Phe346 and Glu372 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 8, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Thr373, Lys369, and Lys343 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 10, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Leu315, Ile371, and Lys343 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 6, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Ile371, Glu372, Lys343, Phe346, and Lys347 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 13, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Glu372 and Thr373 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 15, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

In embodiments of any aspect, the non-covalent interactions comprise an interaction with two or more (such as all of) Lys343, Phe346, and Lys347 of SEQ ID NO: 1. A potent inhibitor of Tau aggregation, compound 18, was identified by the inventors as binding the Tau protein by establishing interactions with the above-mentioned residues, thereby stabilising a conformation of the protein that has reduced propensity to aggregate.

The methods of the first and second aspect may comprise determining whether the candidate compound is able to simultaneously form non-covalent molecular interactions with Lys343 and Glu372 of SEQ ID NO:1.

Multiple compounds found to have a strong effect in modulating Tau aggregation were predicted to form stabilising interactions with Lys343 and Glu372, stabilising the Tau protein in a conformation that include the binding pocket exposing these residues.

In embodiments, a non-covalent molecular interaction with Lys343 comprises a cation-pi interaction and/or a hydrogen bond and/or a pi-H interaction. In some embodiments, a cation-pi interaction with Lys343 is between an aromatic ring of the candidate compound and the sidechain amino group of Lys343. In some embodiments, a hydrogen bond with Lys343 is between an acceptor group of the candidate compound and the backbone amino group of Lys343. In some embodiments, a hydrogen bond with Lys343 is between a donor group of the candidate compound and the backbone carbonyl of Lys343. In some embodiments, a pi-H interaction with Lys343 is between an aromatic ring of the candidate compound and the sidechain Cp of Lys343. In some embodiments, a pi-H interaction with Lys343 is between an aromatic ring of the candidate compound and the backbone amino group of Lys343. In some embodiments, a pi-H interaction with Lys343 is between an aromatic ring of the candidate compound and the sidechain CE of Lys343.

In embodiments, a non-covalent interaction with Ser352 comprises a pi-H interaction. In some such embodiments, a pi-H interaction with Ser352 is between an aromatic ring of the candidate compound and the backbone carbonyl of Ser352.

In embodiments, the non-covalent interaction with any of Leu315, Ser341, Phe346, Lys347, Ile371, Glu372, and Thr373, of SEQ ID NO:1 is a hydrogen bond.

In embodiments, the non-covalent interaction with Glu372 is a hydrogen bond. In some such embodiments, a hydrogen bond with Glu372 is between a H donor group of the candidate compound and the sidechain carboxyl (OE1) of Glu372. In some embodiments, a hydrogen bond with Glu372 is between an acceptor group of the candidate compound and the backbone amino group of Glu372. In some embodiments, a hydrogen bond with Glu372 is between an acceptor group of the candidate compound and the sidechain amino group of Glu372.

In embodiments, the non-covalent interaction with Lys347 is a hydrogen bond. In embodiments, the hydrogen bond with Lys347 is between an acceptor group of the candidate compound and the backbone amino group of Lys347.

In embodiments, the non-covalent interaction with Thr373 is a hydrogen bond. In embodiments, a hydrogen bond with Thr373 is between a donor group of the candidate compound and the hydroxyl group of the side-chain of Thr373.

In embodiments, the non-covalent interaction with Leu315 is a hydrogen bond. In embodiments, a hydrogen bond with Leu315 is between a donor group of the candidate compound and the backbone carbonyl of Leu315.

In embodiments, the candidate compound is able to form a non-covalent interaction with Phe378. In embodiments, the non-covalent interaction is a pi-interaction.

In embodiments, the non-covalent interaction with Ile371 is a hydrogen bond. In embodiments, a hydrogen bond with Ile371 is between an acceptor group of the candidate compound and the backbone Ca of Ile371.

In embodiments, the non-covalent interaction with Ser341 is a hydrogen bond. In embodiments, a hydrogen bond with Ser341 is between an acceptor group of the candidate compound and the sidechain Cp of Ser341.

In embodiments, the non-covalent interaction with Phe346 is a hydrogen bond. In embodiments, a hydrogen bond with Phe346 is between an acceptor group of the candidate compound and the backbone Ca of Phe346.

In embodiments, the non-covalent interaction with Glu342 is a hydrogen bond. In some embodiments, a hydrogen bond with Glu342 is between an acceptor group of the candidate compound and the backbone amino group of Glu342.

In embodiments, the non-covalent interaction with Lys369 is a hydrogen bond. In embodiments, a hydrogen bond with Lys369 is between an acceptor group of the candidate compound and the backbone CE of Lys369. In embodiments, a hydrogen bond with Lys369 is between an acceptor group of the candidate compound and the backbone amino group of Lys369.

In embodiments, the interactions with any of Val350, Leu315, Ile354, Ile371, Phe378 and Phe346 are hydrophobic interactions.

In embodiments, the compound is for inhibiting the aggregation of a Tau protein or a truncated form thereof into paired helical filaments.

The compound may be a small molecule, a peptide, a polypeptide or a combination thereof. Preferably, the compound is a small molecule.

In embodiments, the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 comprises a binding pocket that is capped by Phe378 and Phe346, exposes the hydrophobic side chains of residues Val350, Leu315, Ile354 and/or Ile371, and contains residues Leu315, Lys343, Lys347, Glu372, and/or Thr373 capable of forming hydrogen bonds to a molecule within the binding pocket.

In embodiments, the candidate compound is able to simultaneously form non-covalent molecular interactions with one or more of residues Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Ser352, Lys369, Ile371, Glu372, and Thr373, of SEQ ID NO:1, or equivalent amino acids in a variant or derivative, without exposing hydrophilic groups at a distance below 4 Å from the hydrophobic side chains of residues Val350, Leu315, Ile354 and Ile371.

In embodiments, the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 comprises a hairpin loop between residues Val337 and Gly355 of SEQ ID NO:1.

The present inventors have identified that a compact folded state that is able to interact with inhibitors of Tau aggregation such as LMT, and is stabilised by this interaction, comprises a hair pin loop between residues Val337 and Gly355 of SEQ ID NO:1.

In embodiments, the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 comprises the sequence Pro364-Gly367 located between a loop formed by the sequence Tyr219-Lys331 and the sequence Pro332-Gly335.

In embodiments, the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 is stabilised at least in part by hydrogen bonds between Glu342 and Val318 and/or Thr319, between Gly367 and Lys340, between Asn368 and Lys340, between Lys369 and Glu372, between Lys370 and Asp358, between Ile371 and Ser316, between Glu372 and Ser356, between Thr373 and Gln351, Ser316, between His374 and Gln351, and between Lys375 and Gln351.

The three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 may be that represented by the structure co-ordinates in Table 1, or a structure modelled on these coordinates.

The three-dimensional structure of the part of the Tau protein may be obtainable by performing molecular dynamics simulations of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 in the presence of LMT to obtain one or more complex conformations and selecting a three-dimensional structure of the part of the Tau protein by applying a stability criterion and a binding affinity criterion to the one or more complex conformations.

Advantageously, the stability criterion may apply to the distance between complex conformations in consecutive frames of the molecular dynamics simulation after a predetermined amount of time, and/or the binding affinity criterion may apply to the value of a placement scoring function <−80 kcal/mol or a scoring function <−8.5 as determined by the GB IV scoring function within the CCG MOE docking software.

In embodiments, the stability criterion applies to the root-mean-square deviation (RMSD) of atomic positions, for atoms in the backbone of the part of protein in the complexes, between consecutive frames of the molecular dynamics simulation.

In embodiments, the stability criterion applies to the root-mean-square deviation (RMSD) of atomic positions, for atoms in LMT. In embodiments, a stability criterion may be defined as a threshold on the average RMSD of atomic positions, for atoms in the backbone of the part of protein in the complexes, between consecutive frames of the molecular dynamics simulation, where the average is calculated over a predetermined set of frames of a MD simulation. For example, the predetermined set of frames may be defined as the last 10% of frames, the last 5% of frames, or the last 1% of frames in a MD simulation. The predetermined set of frames may be defined as the last 30 k frames, the least 25 k frames, the last 20 k frames, the last 15 k frames, or the last 10 k frames. The threshold on the average RMSD may be chosen as about 0.5 Å, about 0.6 Å, about 0.7 Å, about 0.8 Å or about about 0.9 Å.

In particular, the present inventors have found that a stable LMT-bound conformation could be obtained by running molecular dynamics simulation of a part of the Tau protein in the presence of LMT, and excluding those complexes where after a predetermined amount of simulation time, the LMT was no longer tightly bound (as indicated by the docking score or ligand RMSD fluctuations) and/or the RMSD fluctuations between consecutive frames of the simulation indicated that the conformation was not stable.

In embodiments, the predetermined amount of time is at least 50 ns, at least 100 ns, or about 100 ns.

In embodiments, the methods described herein further comprises designing a pharmacophore model, wherein the pharmacophore includes features representative of non-covalent molecular interactions with two or more of: Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350, Ser352, Ile354, Lys369, Ile371, Glu372, Phe378 and Thr373, of SEQ ID NO:1.

In embodiments, designing a pharmacophore model comprises computing the interaction energy between the three-dimensional structure of each candidate compound in a library of candidate compounds and the three-dimensional structure of at least a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or a variant or derivative thereof that is structurally equivalent to said region, selecting a subset of the library that has an interaction energy below a threshold, and deriving a pharmacophore model based on the selected subset.

The part of the Tau protein may comprise amino acids 306-378 of SEQ ID NO:1. The part of the Tau protein may comprise amino acids 297-391 of SEQ ID NO:1. The part of the Tau protein may comprise amino acids 295-391 of SEQ ID NO:1. The part of the Tau protein may consist of amino acids 297-391 of SEQ ID NO:1. The part of the Tau protein may consist of amino acids 295-391 of SEQ ID NO:1. The part of the Tau protein may consist of amino acids 306-378 of SEQ ID NO:1. In embodiments, the part of the Tau protein comprises amino acids 306-378 of SEQ ID NO:1 (also referred to herein as dGAE73, SEQ ID NO: 4) or a variant or derivative thereof that is structurally equivalent to said part.

In embodiments, the part of the Tau protein comprises amino acids 297-391 of SEQ ID NO:1 (also referred to herein as dGAE, SEQ ID NO: 3) or a variant or derivative thereof that is structurally equivalent to said part. In embodiments, the part of the Tau protein comprises amino acids 295-391 of SEQ ID NO:1 (also referred to herein as Tau97, SEQ ID NO: 5) or a variant or derivative thereof that is structurally equivalent to said part.

In embodiments, the part of the Tau protein comprises the full sequence of the human Tau isoform 2N4R (SEQ ID NO: 1) or a homolog or variant thereof. In embodiment, a variant has at least 90% amino acid identity with the sequence of SEQ ID NO: 1, or the portion thereof that is represented in the three-dimensional structure.

The methods may further comprise repeating the steps of comparing and determining with a further candidate compound that differs from the previous candidate compound in at least one substituent.

In embodiments, one or more candidate compounds may be modelled such that their properties can be compared to thereby define the features that advantageously stabilise the ligand-bound conformation of the part of the Tau protein. Comparing the properties of multiple candidate compounds may comprise comparing, using computational molecular modelling means, the characteristics of the complexes comprising the part of the Tau protein and each of the candidate compounds in terms of e.g. stability, binding affinity, etc. Comparing the properties of multiple candidate compounds may comprise performing one or more experiments to quantify the Tau aggregation modulation associated with each candidate compound.

In embodiments, such comparisons may be performed sequentially to progressively optimise a candidate compound, where the information obtained by comparing a candidate compound to a previously obtained candidate compound is used to design a new candidate compound in a subsequent iteration. Instead or in addition to this, such comparisons may be performed in parallel where multiple candidate compounds are simultaneously compared to derive insights from common properties of subsets of candidate compounds tested. Such insights can be used to design one or more further candidate compounds.

Without wishing to be bound by theory, it is believed that such a process may help to define the features of a candidate compound that optimally modulates Tau aggregation by stabilising a compact folded conformation of the Tau protein.

Comparing the three-dimensional structure of a candidate compound with a three-dimensional structure of at least a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or a variant or derivative thereof that is structurally equivalent to said region may comprise computing the interaction energy between the candidate compound and the part of the Tau protein represented in the three-dimensional structure.

According to a further aspect, there is provided a computer-implemented method for evaluating the ability of a candidate compound to bind to a binding pocket of a Tau protein, the method comprising the steps of:

-   -   (a) receiving the three-dimensional structure coordinates of a         part of the Tau protein comprising amino acids 315-378 of SEQ ID         NO:1 or equivalent amino acids in a variant or derivative         thereof that is structurally equivalent to said part, wherein         the three-dimensional structure of amino acids 315-378 of SEQ ID         NO: 1 comprises the binding pocket of the Tau protein;     -   (b) performing a fitting operation between a candidate compound         and the binding pocket; and     -   (c) analysing the results of the fitting operation to determine         whether the candidate compound is able to bind to the binding         pocket.

In embodiments, step (c) comprises determine whether the candidate compound is able fit at least in part within the binding pocket and form non-covalent molecular interactions with one or more of Lys343, and at least one of Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350, Ser352, Ile354, Lys369, Ile371, Glu372, Phe378 and Thr373, of SEQ ID NO:1, or equivalent amino acids in a variant or derivative.

In embodiments, the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 is that represented by the structure co-ordinates shown in Table 1, or a structure modelled on these coordinates.

In embodiments, the compound is a small molecule, a peptide, a polypeptide or a combination thereof.

In embodiments, a candidate compound is considered able to bind to the binding pocket if it is able to establish non-covalent interactions with amino acids in the binding pocket. In embodiments, the non-covalent interactions are selected from: hydrophobic interactions, hydrogen bonds, pi-cation interactions, and pi-stack interactions.

In embodiments, a candidate compound that is considered able to bind to the binding pocket is predicted to inhibit the aggregation of the Tau protein or a truncated form thereof. In embodiments, a candidate compound that is considered able to bind to the binding pocket is predicted to inhibit the aggregation of the Tau protein or a truncated form thereof into paired helical filaments.

In embodiments, the method further comprises evaluating the ability of a further candidate compound to bind the Tau protein at a different site from the binding site of the first candidate compound.

In embodiments, evaluating the ability of a further candidate compound to bind the Tau protein at a different site from the binding site of the first candidate compound comprises computing the interaction energy between the further candidate compound and the three-dimensional structure of the part of the Tau protein and determining whether the interaction between the further candidate compound and the Tau protein further stabilises the conformation of the complex between the first candidate compound and the Tau protein.

In embodiments, the first candidate compound is a small molecule or a peptide, and the further candidate compound is a peptide or a polypeptide, such as an antibody or fragment thereof.

In embodiments, the candidate compound is able to bind to the pocket if it is able to simultaneously form non-covalent molecular interactions with two or more of Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350, Ser352, Ile354, Lys369, Ile371, Glu372, Phe378 and Thr373, of SEQ ID NO:1.

In embodiments, the candidate compound is able to bind to the pocket if it is able to simultaneously form non-covalent molecular interactions with Lys343 and at least one of Leu315, Lys347, Glu372 and Thr373.

In some cases, the candidate compound is able to bind to the pocket if it is able to simultaneously form non-covalent molecular interactions with Lys343 and Glu372 of SEQ ID NO:1.

In embodiments, the binding pocket is capped by Phe378 and Phe346, exposes the hydrophobic side chains of residues Val350, Leu315, Ile354 and/or Ile371, and contains residues Leu315, Lys343, Lys347, Glu372, and/or Thr373 capable of forming hydrogen bonds to a molecule within the binding pocket.

In embodiments, the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 comprises a hairpin loop between residues Val337 and Gly355 of SEQ ID NO:1.

In embodiments, the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 comprises the sequence Pro364-Gly367 located between a loop formed by the sequence Tyr219-Lys331 and the sequence Pro332-Gly335.

In embodiments, the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 is stabilised at least in part by hydrogen bonds between Glu342 and Val318, Thr319, between Gly367 and Lys340, between Asn368 and Lys340, between Lys369 and Glu372, between Lys370 and Asp358, between Ile371 and Ser316, between Glu372 and Ser356, between Thr373 and Gln351, Ser316, between His374 and Gln351, between Lys375 and Gln351.

In embodiments, the three-dimensional structure coordinates of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or equivalent amino acids in a variant or derivative thereof that is structurally equivalent to said part have been obtained by performing molecular dynamics simulations of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 in the presence of LMT to obtain one or more complex conformations and selecting a complex conformation using a stability criterion and a binding affinity criterion.

In embodiments, step (a) receiving the three-dimensional structure coordinates of a part of the Tau protein comprises obtaining the three-dimensional structure coordinates of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or equivalent amino acids in a variant or derivative thereof that is structurally equivalent to said part by:

-   -   (a) performing molecular dynamics simulations of a part of the         Tau protein comprising amino acids 315-378 of SEQ ID NO:1 in the         presence of LMT to obtain one or more complex conformations that         differ in their three-dimensional conformations; and     -   (b) selecting a complex conformation using a stability criterion         and a binding affinity criterion, wherein the three-dimensional         structure coordinates of a part of the Tau protein comprising         amino acids 315-378 of SEQ ID NO:1 are defined as the         three-dimensional structure coordinates of the part of the Tau         protein in the selected complex conformation;     -   optionally wherein the stability criterion applies to the         distance between conformations in consecutive frames of the         molecular dynamics simulation after a predetermined amount of         time, and/or wherein the binding affinity criterion applies to         the value of a docking score.

In embodiments, selecting a complex conformation using a stability criterion comprises:

-   -   computing the root-mean-square deviation (RMSD) of atomic         positions, for atoms in the backbone of the part of protein in         the complex conformations, between consecutive frames of the         molecular dynamics simulation over a predetermined amount of         time; and     -   selecting the complex conformation(s) that has/have a RMSD below         a predetermined threshold and/or that have the lowest RMSD         amongst the one or more complex conformations.

In embodiments, the predetermined amount of time is at least 50 ns, at least 100 ns, or about 100 ns.

In embodiments, selecting a complex conformation using a binding affinity criterion comprises computing the interaction energy between the candidate compound and the part of the Tau protein represented in the three-dimensional structure.

In embodiments, performing molecular dynamics simulations of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 in the presence of LMT comprises obtaining the three-dimensional structure coordinates of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or equivalent amino acids in a variant or derivative thereof that is structurally equivalent to said part, wherein the three-dimensional structure coordinates correspond to a conformation of the Tau protein as part of a PHF stack. In embodiments, the three-dimensional structure coordinates correspond to those of PDB ID: 5O3L or a structure modelled on this structure.

The part of the Tau protein may comprise amino acids 306-378 of SEQ ID NO:1. The part of the Tau protein may comprise amino acids 297-391 of SEQ ID NO:1. The part of the Tau protein may comprise amino acids 295-391 of SEQ ID NO:1. The part of the Tau protein may consist of amino acids 297-391 of SEQ ID NO:1. The part of the Tau protein may consist of amino acids 295-391 of SEQ ID NO:1. The part of the Tau protein may consist of amino acids 306-378 of SEQ ID NO:1.

In embodiments, the part of the Tau protein comprises amino acids 306-378 of SEQ ID NO:1 (also referred to herein as dGAE73, SEQ ID NO: 4) or a variant or derivative thereof that is structurally equivalent to said part.

In embodiments, the part of the Tau protein comprises amino acids 297-391 of SEQ ID NO:1 (also referred to herein as dGAE, SEQ ID NO: 3) or a variant or derivative thereof that is structurally equivalent to said part.

In embodiments, the part of the Tau protein comprises amino acids 295-391 of SEQ ID NO:1 (also referred to herein as Tau97, SEQ ID NO: 5) or a variant or derivative thereof that is structurally equivalent to said part.

In embodiments, the part of the Tau protein comprises the full sequence of the human Tau isoform 2N4R (SEQ ID NO: 1) or a homolog or variant thereof. In embodiment, a variant has at least 90% amino acid identity with the sequence of SEQ ID NO: 1, or the portion thereof that is represented in the three-dimensional structure.

In embodiments, the three-dimensional structure coordinates of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or equivalent amino acids in a variant or derivative thereof that is structurally equivalent to said part correspond to a conformation that has the following structural characteristics:

-   -   hydrogen bonds between Glu342 and Val318 and/or Thr319;         optionally wherein the hydrogen bonds are between the Glu342         carboxylic acid and the backbone NH of Val318 and the sidechain         OH of Thr319;     -   one or more hydrogen bonds between one or more of residues         Lys369-Thr377 and one or more of residue Ser341-Gln351;         optionally wherein the one or more bonds comprise:         -   (i) a bond between Gln351 and Thr373, preferably wherein the             bond is between the backbone carbonyl of Gln351 and the             hydroxyl sidechain of Thr373;         -   (ii) a bond between Gln351 and His374, preferably wherein             the bond is between the sidechain carbonyl of Gln351 and the             sidechain amine of His374;         -   (iii) a bond between Gln351 and Lys375, preferably wherein             the bond is between the sidechain carbonyl of Gln351 and the             backbone amine of Lys375;         -   (iv) a bond between Arg349 and Thr377, preferably wherein             the bond is between a sidechain amine of Arg349 and the             hydroxyl sidechain of Thr377, between a sidechain amine of             Arg349 and the hydroxyl backbone of Thr377, and/or between             the carbonyl backbone of Arg349 and the hydroxyl sidechain             of Thr377;         -   (v) a bond between Glu372 and Ser356, preferably wherein the             bond is between the carboxylic acid side chain of Glu372 and             the backbone NH of Ser356, or between the carboxylic acid             side chain of Glu372 and the OH-sidechain of Ser356; and/or         -   (vi) a bond between Glu372 and Lys369, preferably wherein             the bond is between the carboxylic acid side chain of Glu372             and the NH sidechain of Lys369;     -   no beta sheets;     -   a hairpin loop comprising residues Val337-Gly355;     -   the PGGG sequence formed by residues Pro364-Gly367 is within a         distance of 13 A of the PGGG sequence Pro332-Gly335 and/or         within a distance of 2 A of a loop formed by the sequence         Thr319-Lys331;     -   the PGGG sequence formed by residues Pro364-Gly367 is located         between the PGGG sequence Pro332-Gly335 and a loop formed by the         sequence Thr319-Lys331;     -   residues Lys369-Thr377 are within a distance of 6 A of residues         Asp314-Ser316, optionally wherein the distance between the         Ser316 beta-carbon and Thr373 backbone carbonyl is between 2.5 Å         and 5.0 Å;     -   residues Gly355-Gly367 and Asn368-Arg379 are within 2 A hydrogen         bonding distance;     -   Glu338 is folded towards Val363, optionally wherein the distance         (RMSD) between the carbonyl oxygen side chain of Glu338 and the         backbone amine nitrogen of Val363 NH during the final 10 ns of a         50 ns simulation is between 2 Å and 4 Å, or below 5 Å;     -   the total water accessible surface area calculated for the part         of the protein is at least 20% lower than the corresponding         values calculated for a conformation as provided by the         three-dimensional coordinates with PDB identifier 5O3L; and/or     -   the polar and/or hydrophobic accessible surface area(s)         calculated for the part of the protein is/are at least 20% lower         than the corresponding values calculated for a conformation as         provided by the three-dimensional coordinates with PDB         identifier 5O3L.

In embodiments, the three-dimensional structure coordinates of the part of the Tau protein is such that the distance (RMSD) between the backbone carbonyl oxygen of Gln351 and the sidechain hydroxyl oxygen of Thr373 during the final 10 ns of a 50 ns simulation is between 2 Å and 4 Å, or below 5 Å.

In embodiments, the three-dimensional structure coordinates of the part of the Tau protein is such that the distance (RMSD) between the sidechain carbonyl oxygen of Gln351 and the sidechain amine nitrogen of His374 during the final 10 ns of a 50 ns simulation is between 2 Å and 4 Å, or below 5 Å.

In embodiments, the three-dimensional structure coordinates of the part of the Tau protein is such that the distance (RMSD) between the sidechain carbonyl oxygen of Gln351 and the backbone amine nitrogen of Lys375 during the final 10 ns of a 50 ns simulation is between 2.5 Å and 5 Å, or below 5 Å.

In embodiments, the three-dimensional structure coordinates of the part of the Tau protein is such that the distance (RMSD) between the backbone carbonyl oxygen of Arg349 and the hydroxyl sidechain oxygen of Thr377 during the final 10 ns of a 50 ns simulation is between 2.5 Å and 4 Å, or below 5 Å.

In embodiments, the three-dimensional structure coordinates of the part of the Tau protein is such that the distance (RMSD) between the carboxylic acid sidechain oxygen of Glu372 and the backbone NH of Ser356 during the final 10 ns of a 50 ns simulation is between 2 Å and 4 Å, or below 5 Å.

According to a further aspect, there is provided a method for selecting or designing a compound for modulating the aggregation of a Tau protein or a truncated form thereof, the method comprising:

-   -   using a three-dimensional structural model of at least a part of         the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or         a variant or derivative thereof that is structurally equivalent         to said part, wherein the model is an intermediate in the         aggregation process of the part of the Tau protein with a paired         helical filament (PHF), wherein the model is generated by         simulating the conformational changes of the part of the Tau         protein from a compact folded state to a an aggregated state         such that:         -   (i) residues Val337-Gln355 form a hairpin loop that moves to             align with alternating positively charged and negatively             charged sidechain stacks in the hairpin loop of the PHF;         -   (ii) residue Pro332 switches between a trans and a cis             configuration;         -   (iii) residues 355-378 and 306-318 move to form stabilising             cross-β sheets with corresponding residues of the PHF             through hydrophobic zippering; and generating a model of a             complex of the compound and the intermediate.

In embodiments, residue Pro332 switching between a trans and a cis configuration causes residues His329, His330 and Lys331 to move close enough to PHF to establish interactions with the partner residues in the next layer of the PHF stack through hydrophobic stacking and a strong hydrogen bond between the side chains of His330 in a preformed layer of the PHF stack and Thr361 of the newly formed dGAE layer on the PHF stack.

In embodiments, step (iii) comprises the following steps: (iii)(1) residues 355-360 move to establish a zipper with the corresponding anti-parallel residues of the PHF; (iii)(2) residues 361-367 move to establish a zipper with the corresponding anti-parallel residues of the PHF; and (iii)(3) residues 306-318 and 368-378 move to form a cross-beta sheet conformation with corresponding residues of the PHF.

In embodiments, residues 355-360 move to establish a zipper with the corresponding anti-parallel residues of the PHF from the C- to N-direction.

In embodiments, residues 361-367 move to establish a zipper with the corresponding anti-parallel residues of the PHF in the N- to C-direction.

In embodiments, the cross-beta sheet conformation is formed simultaneously starting from Phe378 and closing the residues from C- to N-terminal, and joining residues 318-306 of the N-terminal in the C- to N-direction.

In embodiments, the compound is a small molecule, a peptide, a polypeptide or a combination thereof. In embodiments, the polypeptide is an antibody or a fragment thereof.

In embodiments, the method further comprises selecting or designing a further compound for modulating the aggregation of a Tau protein or a truncated form thereof by generating a model of a complex of the first compound, the further compound and the intermediate.

In embodiments, the further compound is a small molecule, a peptide, a polypeptide or a combination thereof. In embodiments, the polypeptide is an antibody or a fragment thereof.

The compact folded state may have any of the structural characteristics defined in relation to the preceding aspect.

The compact folded state may have the structure co-ordinates shown in Table 1, or a structure modelled on these coordinates.

Generating a model of a complex of the compound and the intermediate may comprise identifying the compound as binding to the intermediate and preventing the occurrence of any of steps (i) to (v).

Generating a model of a complex of the compound and the intermediate may comprise identifying the compound as binding to the intermediate and preventing the occurrence of any of steps (i) to (iii).

In embodiments, simulating the conformational changes of the part of the Tau protein from a compact folded state to an aggregated state comprises performing a molecular dynamics simulation of the assembly of a Tau protein or a part thereof with a PHF stack. In embodiments, the PHF stack comprises at least 2, at least 4, at least 6, at least 8 or at least 10 monomers. In embodiments, the PHF stack comprises the structure defined in PDB ID: 5O3L, or a structure modelled from the structure in PDB ID: 5O3L.

In embodiments, the part of the Tau protein comprises amino acids 306-378 of SEQ ID NO:1 (also referred to herein as dGAE73, SEQ ID NO: 4) or a variant or derivative thereof that is structurally equivalent to said part.

In embodiments, the part of the Tau protein comprises amino acids 297-391 of SEQ ID NO:1 (also referred to herein as dGAE, SEQ ID NO: 3) or a variant or derivative thereof that is structurally equivalent to said part.

In embodiments, the part of the Tau protein comprises amino acids 295-391 of SEQ ID NO:1 (also referred to herein as Tau97, SEQ ID NO: 5) or a variant or derivative thereof that is structurally equivalent to said part.

In embodiments, simulating the conformational changes of the part of the Tau protein from a compact folded state to an aggregated state comprises performing a first set of molecular dynamics simulation of the assembly of a Tau protein or a part thereof with a PHF stack, in which the anchoring of the part of the Tau protein to the PHF stack is determined by starting the molecular dynamics simulations in the set with different relative orientations of the part of the Tau protein and the PHF stack.

In embodiments, the molecular dynamics simulation in the set are each started with a random orientation of the part of the Tau protein. Preferably, the molecular dynamics simulation in the set are each started with the part of the Tau protein placed beyond hydrogen-bonding distance (such as e.g. at least 4 Å away) of the PHF stack.

The present inventors have found that such molecular dynamics simulation enabled the identification of an intermediate in which the part of the Tau protein has anchored itself on the PHF stack. Such an intermediate may correspond to one where step (i) has occurred.

In embodiments, simulating the conformational changes of the part of the Tau protein from a compact folded state to an aggregated state comprises performing a nudged elastic band molecular dynamics simulation starting from an intermediate in which step (i) has occurred.

According to a further aspect, there is provided a computing system comprising a processor and a memory storing machine-readable instructions that, when executed by the processor, cause the processor to implement the method of any embodiment of the aspects described above.

According to a further aspect, there is provided a computer readable storage medium (or a plurality of computer readable storage media) storing instructions that, when executed by one or more processors, cause the processor(s) to implement the method of any embodiment of the aspects described above.

According to a further aspect, there is provided a computer software product comprising instructions that, when executed by one or more processors, cause the processor(s) to implement the method of any embodiment of the aspects described above.

FIGURES

FIG. 1 . Flowchart illustrating a method for identifying, selecting and/or designing a compound for modulating Tau aggregation according to the present disclosure.

FIG. 2 . Flowchart illustrating a method for identifying, selecting and/or designing a compound for modulating Tau aggregation according to the present disclosure.

FIG. 3 . Schematic illustration of the steps used to analyse the structure of Tau97 (AspAsn-dGAE) monomers and LMT-bound Tau97 (AspAsn-dGAE) monomers.

FIG. 4 . PCA Subspace comparison of 30 molecular trajectories obtained by independent molecular dynamics simulations of Tau97 monomers. A. Hierarchical clustering of PCA subspace of the 30 trajectories. B. Subspace overlap between the 30 molecular trajectories.

FIG. 5 . Heatmap of 178 pairwise RMSD measurements between Tau97 protein conformations derived from the MD sampling (ligand free) and PCA analysis of FIGS. 3-4 .

FIG. 6 . PCA Subspace comparison of 22 molecular trajectories obtained by cycles of molecular dynamics simulations of LMT-bound Tau97 complexes followed by docking rescoring to identify tightly bound complexes.

FIG. 7 . PCA subspace comparison between the 22 molecular trajectories of FIG. 6 and the 30 molecular trajectories of FIG. 4 .

FIG. 8 . LMT-Tau97 pose found to stabilise the monomer Tau97 protein. A. Cartoon representation of Tau97 bound to LMT (sticks representation), the bottom left is the figure rotated by 90 degrees, and also showing the residues within the binding site. Top right is the LMT binding site. Bottom right is a space filling and surface representation of LMT-bound Tau97. The colours correspond to the colours in the primary sequence indicated at the top. B. Plot of the RSMD (root mean square deviation) of the residues surrounding LMT, D₃₁₄-S₃₁₆, E₃₄₂-K₃₅₃, and K₃₇₀-H₃₇₄. Line plot shows the RSMD from the first frame (top curve) and the RSMD from each preceding frame (bottom curve), plotted as the mean for every 200 ps of simulation time. Error bars indicate the standard deviation for every 200 ps of simulation.

FIG. 9 . A-B. Cartoon representations of PHF, residues 306-378 from PDB id 5O3L. C. LMT-Tau97 pose of FIG. 8 with Van der Waals surface coloured by electrostatics, blue is electropositive, and red is electronegative. This figure shows that K343 and K347 form an electropositive cap over the ligand binding site and that Phe378 also caps the pocket with a hydrophobic lid. D. Cartoon representation of the LMT-Tau97 pose of FIG. 8 showing residues within 4 Å of LMT within the binding pocket shown as sticks. E. The LMT binding site in greater detail. The colours on A, B, D, E correspond to the colours in the primary sequence indicated at the bottom of the figure.

FIG. 10 . Distances between heavy atoms pairs along the final 10 ns of a 50 ns simulation for the protein complex of FIG. 8 , and the 30 Tau97 structures of FIG. 4 . A. Gln351 to Thr373. B. Gln351 to His374. C. Gln351 to Lys375. D. Arg349 to Thr377. E. Ser316 to Thr373. F. Ser356 to Glu372. F. Glu338 to Val363.

FIG. 11 . Cartoon representation of the complex of FIG. 8 , highlighting the strong hydrogen bonding features which stabilise the folded state.

FIG. 12 . LMT structure and binding pocket. A-C. Chemical structures of MT (A), LMTM (B) and LMT (C). D. Close up view of the cryptic binding pocket of LMT in the Tau97 conformation of FIG. 8 , with van der Waals surface coloured by hydrophobicity, with ligand (LMT) pharmacophore features indicated as spheres (hydrophobic features: green spheres, aromatic features: orange spheres, hydrogen-bond donor: magenta spheres).

FIG. 13 . Cartoon representation of the approach of a dGAE73 monomer (colour by the standard deviation in position relative to the binding partner in the would-be fully assembled state, spectrum illustrated, from a high of 18.35 Å in red to a low of 0.87 Å in blue) towards a PHF 10 oligomeric stack, cyan cartoon representation. The top picture is from a view horizontal to the PHF stack, and the bottom picture is the same image rotated by 90° towards the reader.

FIG. 14 . Snapshots taken from the molecular dynamics simulations of the anchoring stages in PHF assembly. A) The initial stage showing a bound LMT ligand. B) After 1.2 ns of simulation of dGAE73. C) After 41.2 ns of simulation of dGAE73, when anchoring was deemed to be complete. D) At the start of the unfolding of the dGAE73.

FIG. 15 . Analysis of the dGAE73 folding pathway during PHF assembly. A-F. Frames 1, 81, 101, 139, 142 and 144, respectively, from the assembly stage of the molecular dynamics simulation—the structure in A is the structure shown in FIG. 14D), from a different angle. G. Scatterplots tracking the progress of PHF formation of a monomeric dGAE73 protein: a point on the plot illustrates at what frame in the MD simulation a residue of the monomeric dGAE73 is close to the final position it would assume in a fully assembled PHF. The Y-axis represents the residue numbering from 308-378, and the X-axis if the frame number from the MD simulation; frames 1-144 (from the beginning to the completion of assembly) on the left and frames 131-144 on the right. The amino acid sequence is shown below, coloured according to the linear epitopes of four antibodies used to probe the assembly process (from N- to C-ter: 319-331, 337-355, 355-367, 367-378)—also indicated by dashed lines in the cartoon representation and along the y axis on the right side of each plot.

FIG. 16 . The initial dGAE73 binding before full assembly commences. A. Representation of the top view of one arm of the PHF with the Van der Waals surface coloured according to electrostatic charge (red is electronegative and blue is electropositive). The top left inset is the cartoon representation of the same structure. B. Representation of A, rotated by 90°, the alternating acidic-basic residue wall of the PHF is highlighted.

FIG. 17 . dGAE73 proline 332 switch during PHF assembly. A. Psi and omega dihedral angles of Pro332 observed during the molecular dynamics simulation of dGAE73 assembly onto PHF-transitions from trans-cis-trans are indicated on the plot. B-D. Frames 40, 100 and 138 of the simulation shown as stick representation, with Psi, Psi1 and Omega indicated.

FIG. 18 . Hydrogen bonding interactions in a PHF. A. The hydrogen bonding interactions observed in a single layer of a dGAE73 monomer in a PHF from residues V306-Ser320 and Gly367-Phe378. B. Cross-β sheet formation within PHFs are formed through multiple hydrogen-bonds along the protein backbone.

FIG. 19 . Graphical representation of analysis of densitometry from dot blots with dGAE antibodies during PHF formation. Binding of the antibodies indicates which parts of the sequence fold first. A. Cartoon representation of folded dGAE showing the region that is bound by each antibody used, and the results of exemplary immunoblots. B. Graphical representation of analysis of densitometry from dot blot for antibodies with linear epitopes 306-359, 319-331, 337-355, 355-367, 367-378, 379-390 and 379-391, for first 8 hours of incubation. C. Graphical representation of analysis of densitometry from dot blots for same antibodies as A, showing the full timeline of incubation (0-80 hours).

FIG. 20 . RMSD in Å of the epitopes sequences alone (A) and in the dGAE monomer during the simulated assembly into PHFs (B and C). A. Epitope RMSD using the antibody bound epitope conformation as a point of reference (antibody linear epitopes as indicated). B. RMSD with the final assembled epitope conformation as a point of reference (antibody linear epitopes as indicated). C. RMSD with the starting conformation as a point of reference (antibody linear epitopes as indicated).

FIG. 21 . Sandwich ELISA graphs showing the increase in immunoreactivity of core region scAbs to dGAE ‘total’, ‘supernatant’ and ‘pellet’ aggregation inhibition samples prepared in the presence of LMTM. dGAE monomer was included as assay control to indicate the binding profiles of each test scAbs to their corresponding epitopes in non-aggregated samples. (A-C) AB3 scAb, (D-F) AB8, (G-1) AB5 scAb, (J-L) AB1 scAb, (M-O) AB7, (P-R) AB2 scAb. Lack of antibody binding in some dGAE+LMTM aggregate pellet samples corresponds to the absence protein present in this group as confirmed by SDS gel (data not included).

FIG. 22 . Protease digestion experiments reveal that dGAE filaments contain a protease resistant core (A-B) and that LMT leads to a loss of protease resistance (C). SDS-PAGE gels are shown for the supernatant (A) and pellet (B) following incubation of fibrils of dGAE in reducing conditions with increasing concentrations of proteinase K or Pronase E. The sequence of dGAE is indicated below panel B, with the sequence of the protease resistant core (band at around 8 kDa, revealed by mass spectrometry to correspond to a protected core region of H299-K370 with a theoretical molecular weight of 7.5 kDa) highlighted. C. SDS-PAGE gel showing that in the presence of DTT, LMT is able to prevent inhibition and the resulting soluble dGAE can be digested.

FIG. 23 . Immunogold labelling experiments. A. Immunogold labelling of soluble (left), preformed filaments of dGAE (middle) and soluble capped tau350-362/cappedtau350-362 filaments (right) using an antibody binding the region highlighted in the cartoon representation (358-364) confirm that the recognition sequence of this antibody is exposed in soluble dGAE and buried in PHF. B. Immunogold labelling of dGAE filaments using scAbs targeting the regions highlighted in the cartoon representations show very little labelling and thus reduces further following SDS (to remove the soluble dGAE—framed images).

DETAILED DESCRIPTION

All residue numbers of the Tau protein sequence and structure in the present disclosure refer to the residues of SEQ ID NO:1, which is the sequence of the four repeat isoform 2N4R of human Tau protein (Uniprot ID P10636-8), or homologous positions in other species or variants thereof. Human Tau isoform 2N4R (Uniprot ID P10636-8) corresponds to amino acids 1-124, 376-394 and 461-758 of full length Tau, Uniprot ID P10636 or P10636-1, provided as SEQ ID NO:2.

SEQ ID NO: 1 (Isoform Tau-F, also known as Tau-4, 2N4R, 441 amino acids): >sp|P10636-8|TAU_HUMAN Isoform Tau-F of Microtubule-associated protein tau OS = Homo sapiens OX = 9606 GN = MAPT MAEPROEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPG SETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAG HVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPK TPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAK SRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHV PGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNI THVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMV DSPQLATLADEVSASLAKQGL SEQ ID NO: 2 (Full length human Tau, Isoform PNS-Tau, 758 amino acids); >sp|P10636|TAU_HUMAN Microtubule-associated protein tau OS = Homo sapiens OX = 9606 GN = MAPT PE = 1 SV = 5 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPG SETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAG HVTQEPESGKVVQEGFLREPGPPGLSHQLMSGMPGAPLLPEGPREATRQPSGTGPEDTEG GRHAPELLKHQLLGDLHQEGPPLKGAGGKERPGSKEEVDEDRDVDESSPQDSPPSKASPA QDGRPPQTAAREATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPDGPSVGRAKGQDAPLE FTFHVEITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSLGEDTKEADLPEPSEKQPA AAPRGKPVSRVPQLKARMVSKSKDGTGSDDKKAKTSTRSSAKTLKNRPCLSPKHPTPGSS DPLIQPSSPAVCPEPPSSPKYVSSVTSRTGSSGAKEMKLKGADGKTKIATPRGAAPPGQK GQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREP KKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLD LSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEK LDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDT SPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

References to residues Leu315, Phe346, Lys347, Val350, Ile354, Ile371, Thr373, and Phe378, refer to the residues in bold and underline in the sequence of SEQ ID NO:1 reproduced below (or the corresponding residues in any fragment, homologue or variant thereof, including e.g. dGAE, Tau97 or dGAE73 as defined below):

        10         20         30         40 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD         50         60         70         80 AGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLV         90        100        110        120 DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG        130        140        150        160 HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP        170        180        190        200 GQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSP        210        220        230        240 GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK        250        260        270        280 SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK        290        300        310        320 KLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVD L SKVIS        330        340        350        360 KCGSLGNIHH KPGGGQVEVK SEKLD FK DR V  QSK I GSLDNI        370        380        390        400 THVPGGGNKK  I E T HKLI F RE NAKAKTDHGA EIVYKSPVVS        410        420        430        440 GDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L

dGAE refers to the 95 residues fragment of Tau (2N4R) with N-terminus at residue Ile-297 and C-terminus at residue Glu-391, as described in SEQ ID NO: 3, or at homologous positions in other species (the residues mentioned referring to the human or mouse Tau sequence, which are identical in this region). As will be apparent to the skilled person, dGAE95 also corresponds to the fragment of the large peripheral nervous system (PNS) Tau isoform (P10636-1) with N-ter at Ile-614 and C-ter at Glu-708. This sequence may sometimes be referred to simply as “dGAE”.

SEQ ID NO: 3 (dGAE, human/mouse, 95 amino acids): IKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLD FKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAE

dGAE73 refers to the fragment of Tau (2N4R) with N-terminus at residue Val-306 and C-terminus at residue Phe-378, as described in SEQ ID NO: 4, or at homologous positions in other species (the residues mentioned referring to the human or mouse Tau sequence, which are identical in this region). As will be apparent to the skilled person, dGAE73 also corresponds to the fragment of Isoform PNS-Tau (P10636-1) with N-ter at Val-623 and C-ter at Phe-695.

SEQ ID NO: 4 (dGAE73, human/mouse, 73 amino acids): VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKI GSLDNITHVPGGGNKKIETHKLTF

Tau97 refers to the 97 residues fragment of Tau (2N4R) with N-terminus at residue Asp-295 and C-terminus at residue Glu-391, as described in SEQ ID NO: 5, or at homologous positions in other species (the residues mentioned referring to the human or mouse Tau sequence, which are identical in this region). As will be apparent to the skilled person, Tau97 also corresponds to the fragment of the large peripheral nervous system (PNS) Tau isoform (P10636-1) with N-ter at Asp-612 and C-ter at Glu-708. This sequence may also be referred to as “dGAE97” of “AspAsn-dGAE” as it includes the 95 amino acids dGAE and the two preceding amino acids Asp and Asn.

SEQ ID NO: 5 (Tau97, human/mouse, 97 amino acids): DNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEK LDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAE

In the context of the present disclosure, polypeptides are considered to be “structurally equivalent” if they are able to fold into conformations that have the same three-dimensional interaction properties. In particular, a polypeptide that folds into at least one conformation that has a binding pocket is structurally equivalent to another polypeptide if the latter folds into at least one conformation that has a binding pocket, where the binding pockets of the two polypeptides can be stabilised by forming molecular interactions with the same ligand at corresponding positions in the ligand and the amino acid sequences of the respective polypeptides. For example, two polypeptides may be structurally equivalent if the primary sequence of the first polypeptide differs from the primary sequence of the second polypeptides such that the two polypeptides have at least one predicted conformation that forms a binding pocket, and the interaction properties of the binding pocket are not materially affected by the difference in primary sequence. This may be the case for example where one or more substitutions are made that do not change the conformation of the binding pocket and that do not affect residues that establish stabilising interactions with the ligand, or do not affect said residues in such a way that the stabilising interaction is no longer present. In particular, a variant of dGAE (or Tau97 or dGAE73) may be considered to be structurally equivalent to dGAE (or Tau97 or dGAE73) if it is able to fold in a conformation that forms a binding pocket that comprises multiple hydrophobic side chains and is able to accommodate LMT in a pose where LMT forms stabilising interactions with the amino acids in the variant equivalent to Lys343 and Thr373 of dGAE (or Tau97 or dGAE73).

Aggregation of the tau protein is a hallmark of diseases referred to as “tauopathies”. Various tauopathy disorders that have been recognized which feature prominent tau pathology in neurons and/or glia and this term has been used in the art for several years. The similarities between these pathological inclusions and the characteristic tau inclusions in diseases such as AD indicate that the structural features are shared and that it is the topographic distribution of the pathology that is responsible for the different clinical phenotypes observed. In particular, cryo-electron microscope structures of aggregated Tau in AD, Frontotemporal dementia (including Pick's disease), chronic traumatic encephalopathy (CTE) and cortico-basal degeneration (CBD) have been previously obtained, and all show common conformational features, indicating that compounds that have the ability to modulate Tau aggregation in e.g. PHFs (as observed in AD), may also modulate aggregation of Tau in other tauopathies. In addition to specific diseases discussed below, those skilled in the art can identify tauopathies by combinations of cognitive or behavioural symptoms, plus additionally through the use of appropriate ligands for aggregated tau as visualised using PET or MRI, such as those described in WO02/075318.

Aspects of the present invention relate to “tauopathies”. As well as Alzheimer's disease (AD), the pathogenesis of neurodegenerative disorders such as Pick's disease and Progressive Supranuclear Palsy (PSP) appears to correlate with an accumulation of pathological truncated tau aggregates in the dentate gyrus and stellate pyramidal cells of the neocortex, respectively. Other dementias include fronto-temporal dementia (FTD); parkinsonism linked to chromosome 17 (FTDP-17); disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC); pallido-ponto-nigral degeneration (PPND); Guam-ALS syndrome; pallido-nigro-luysian degeneration (PNLD); cortico-basal degeneration (CBD); Dementia with Argyrophilic grains (AgD); Dementia pugilistica (DP) wherein despite different topography, NFTs are similar to those observed in AD (Bouras et al., 1992); Chronic traumatic encephalopathy (CTE), a tauopathy including DP as well as repeated and sports-related concussion (McKee, et al., 2009). Others are discussed in Wischik et al. 2000, for detailed discussion—especially Table 5.1).

Abnormal tau in NFTs is found also in Down's Syndrome (DS) (Flament et al., 1990), and in dementia with Lewy bodies (DLB) (Harrington et al., 1994). Tau-positive NFTs are also found in Postencephalitic parkinsonism (PEP) (Charpiot et al., 1992). Glial tau tangles are observed in Subacute sclerosing panencephalitis (SSPE) (Ikeda et al., 1995). Other tauopathies include Niemann-Pick disease type C (NPC) (Love et al., 1995); Sanfilippo syndrome type B (or mucopolysaccharidosis Ill B, MPS Ill B) (Ohmi, et al., 2009); myotonic dystrophies (DM), DM1 (Sergeant, et al., 2001 and references cited therein) and DM2 (Maurage et al., 2005). Additionally there is a growing consensus in the literature that a tau pathology may also contribute more generally to cognitive deficits and decline, including in mild cognitive impairment (MCI) (see e.g. Braak, et al., 2003, Wischik et al., 2018).

All of these diseases, which are characterized primarily or partially by abnormal tau aggregation, are referred to herein as “tauopathies” or “diseases of tau protein aggregation”. In aspects of the invention relating to tauopathies, preferably the tauopathy is selected from the list consisting of the indications above, i.e., AD, PSP, FTD (including Pick's disease), FTDP-17, DDPAC, PPND, Guam-ALS syndrome, PNLD, and CBD and AgD, DS, SSPE, DP, PEP, DLB, CTE and MCI. In one preferred embodiment the tauopathy is Alzheimer's disease (AD). Without wishing to be bound by theory, the present inventors believe that all structures solved for tauopathies encompass the dGAE region of Tau. As such, findings in relation to stabilising a conformation of dGAE (or Tau97) that is not prone to assembly can reasonably be expected to apply to all tau diseases including but not limited to AD.

Aspects of the present disclosure relate to methods for identifying, selecting and/or designing a compound for modulating Tau aggregation, which make use of computer-implemented molecular modelling means.

As illustrated in FIG. 1 , the method may comprise steps of receiving or obtaining 110 the structure coordinates of a part of the Tau protein as described herein, comparing 120 the three-dimensional structure of a candidate compound with the three-dimensional structure of at least a part of the Tau protein (such as e.g. by performing a fitting operation between a candidate compound and a binding pocket in the Tau protein as described herein), and analysing 130 the results to determine whether the candidate compound is able to bind to the binding pocket. Analysing the results of the fitting operation may comprise determining whether the candidate compound is able fit at least in part within the binding pocket and form non-covalent molecular interactions with one or more of Lys343, and at least one of Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350, Ser352, Ile354, Lys369, Ile371, Glu372, Phe378 and Thr373, of SEQ ID NO:1, or equivalent amino acids in a variant or derivative. Obtaining the structure coordinates of a part of the Tau protein may comprise receiving the structure coordinates of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 in a PHF stack (such as e.g. from PDB ID: 5O3L), performing molecular dynamics simulations of a part of the Tau protein in the presence of an inhibitor of Tau aggregation as described herein (such as e.g. LMT) and selecting an inhibitor-bound complex conformation using a stability criterion and a binding affinity criterion. Further, performing molecular dynamics simulations may comprise performing a set of independent molecular simulations to obtain a set of molecular trajectories, performing PCA analysis of the molecular trajectories to identify a set of representative conformations, and performing molecular dynamics simulations of a part of the Tau protein in the presence of an inhibitor of Tau aggregation as described herein (such as e.g. LMT) using each of the representative conformations.

As illustrated on FIG. 2 , the method may alternatively comprise steps of receiving or obtaining 210 the structure coordinates of a part of the Tau protein, wherein the structure coordinates are those of an intermediate in the aggregation process of the part of the Tau protein with a PHF, and generating 220 a model of a candidate compound and Tau protein. Obtaining 210 the structure coordinates of a part of the Tau protein, wherein the structure coordinates are those of an intermediate in the aggregation process of the part of the Tau protein with a PHF, may comprise simulating 212 the conformational changes of the part of the Tau protein from a compact folded state to an aggregated state by performing a molecular dynamics simulation of the assembly of a Tau protein or a part thereof with a PHF stack. The compact folded state may have been obtained as described above in relation to step 110. In particular, the structure coordinates of a part of the Tau protein in a compact folded state may have been obtained by receiving the structure coordinates of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 in a PHF stack (such as e.g. from PDB ID: 5O3L), performing molecular dynamics simulations of a part of the Tau protein in the presence of an inhibitor of Tau aggregation as described herein (such as e.g. LMT) and selecting an inhibitor-bound complex conformation using a stability criterion and a binding affinity criterion. Further, performing molecular dynamics simulations may comprise performing a set of independent molecular simulations (e.g. starting from the coordinates in PDB ID: 5O3L or a structure modelled form those coordinates) to obtain a set of molecular trajectories, performing PCA analysis of the molecular trajectories to identify a set of representative conformations, and performing molecular dynamics simulations of a part of the Tau protein in the presence of an inhibitor of Tau aggregation as described herein (such as e.g. LMT) using each of the representative conformations. Alternatively, the structure coordinates of a part of the Tau protein in a compact folded state may have been obtained by performing a set of independent molecular simulations (e.g. starting from the coordinates in PDB ID: 5O3L or a structure modelled form those coordinates) to obtain a set of molecular trajectories, performing PCA analysis of the molecular trajectories to identify a set of representative conformations, and selecting one or more representative conformations as a compact folded state.

Candidate compounds may be selected using in silico methods known in the art. For example, in silico methods via substructure search may be employed. The structure of a known modulator of Tau aggregation, such as e.g. LMT, may be used, and various structural features of the compound (such as e.g. hydrophobic features, H-bond acceptor or donor features, etc.) may be submitted to a program that will search through libraries of chemical compounds for chemicals with substructures that have similar features. The candidate structures may then optionally be reviewed by an expert, for example in order to remove those compounds that are too large. Selected candidates may be submitted to a program that creates structural coordinates for the compounds, such as e.g. AMBER. Docking and scoring of these candidates may be performed using e.g. the MOE software from CCG.

Selected or designed compounds may further be synthesised or obtained and tested for their ability to modulate Tau aggregation, for example in a cell-based aggregation assay as described in Rickard et al., 2017.

Molecular dynamics simulations may be performed as known in the art, for example using the tools available as part of the AMBER software.

EXAMPLES Example 1—Analysis of the Structure of dGAE Monomers

Overview

In this example, the conformational landscape of dGAE monomers in an aqueous environment was studied using explicit water molecular dynamics simulations based on the model from Fitzpatrick et al. (2017)—from PDB identifier 5O3L (https://www.rcsb.org/structure/5O3L). In particular, a slightly extended version of dGAE (Tau97) was used. However, the results herein are believed to be unaffected by the presence of 2 additional N-terminal residues, and equally apply to dGAE. As such, any references to “Tau97” in this example apply equally to dGAE.

Methods

Protein molecular dynamics (MD) simulations (see FIG. 3 , Step 310): 3D protein structures of PDB ID: 5O3L (Fitzpatrick et al., 2017) was obtained from the PDB (rcsb.org, Berman et al., 2000). The coordinates of atoms were extracted from the files and only single monomers were used from crystal structures that contained dimers. The sequence utilised in the modelling consisted of Tau97, and refers to the 97-residue fragment of Tau (2N4R) with N-terminus at residue Asp-295 and C-terminus at residue Glu-391 (i.e. comprising the dGAE fragment with N-terminus at residue Asp-297 and C-terminus at residue Glu-391). All the waters were removed from the structures. Protein parameterisation was performed using the MOE 2016.0802 software package (The Molecular Operating Environment, http://www.chemcomp.com).

The structural model was then refined using an energy minimisation process, by means of molecular mechanics using the AMBER force field. Proteins were parameterised in LEaP (a module from the Amber suite of programmes which generates force field files for use with the molecular dynamics packages of Amber) using the AMBER ff14SB force field (Maier et al., 2015). The complexes were neutralised by addition of an appropriate number of chloride counter ions and immersed in a truncated octahedral box of pre-equilibrated TIP3P water molecules (Jorgensen, 1982; Jorgensen et al., 1983). Each water box extended 8 Å away from any solute atom. At each stage, parameter, topology and coordinate files were saved. The cut-off distance for non-bonded interactions was 10 Å. Periodic boundary conditions were applied and electrostatic interactions were represented using the smooth PME method (smooth particle mesh Ewald method; Darden et al., 1998), with constant volume conditions applied. Minimisations were performed using the sander module (MD simulation engine) of AMBER 17 package (Case et al., 2017). The simulation protocol involved initial solvent and ion density equilibration and minimisation. A total of 2,000 minimisation steps were performed; initially 1,000 steps of steepest descent, followed by 1,000 steps of conjugate gradient minimisation. The cut-off distance for non-bonded interactions was 10 Å and a force constant of 500 kcal/mol/Å² was used to restrain the protein. The entire system was then subjected to 2,500 steps of minimisation without restraints.

Following protein refinement using energy minimisation, a 200 ps heating phase with a cut-off distance for non-bonded interactions of 10 Å and a force constant of 10 kcal/mol/Å² was used to restrain the protein, before a 200 ps equilibration run. At this point, 30 independent replicas were generated by randomly assigning different sets of velocities (adjusted to a temperature of 300 K) to the initial coordinates.

For each replica a 60 ns unrestrained trajectory was simulated at 300 K temperature and 1 atm pressure. SHAKE (Ryckaert et al., 1977; a constraint algorithm that can be applied to molecular dynamics simulations to ensure that constraints such as bond length, bond angle and torsion angle constraints are satisfied—in this case the algorithm constrains the vibrational stretching of hydrogen bond lengths, fixing the bond distance to equilibrium value) was applied to all bonds involving hydrogen atoms, allowing an integration step of 2 fs. System coordinates were saved every 100 ps for further analysis.

Analysis of conformational flexibility (FIG. 3 , Step 320): Principal Component Analysis (PCA) was used to examine the conformational variability amongst the 30 independent simulations. Analysis of trajectories was done with the cpptraj module of AMBER (Roe and Cheatham, 2013) and a locally modified version of pyPCAzip package (Shkurti et al., 2016). A total of 25,000 frames were extracted from each simulation replica. These frames were evenly taken over the last 50 ns of the trajectories, totally 750,000 frames over the 30 replicas. PCA is commonly used to extract the larger amplitude motions that can be observed in the MD trajectories. A subspace (set of eigenvectors obtained by diagonalization of the covariance matrix, giving a vectorial description of components of the motion) can be derived from a PCA analysis of each trajectory, and these can be compared between trajectories in a simple manner.

Results

A total of 30 independent 50 ns molecular simulations were set up and analysed using statistical methods to see whether protein folds followed a similar trajectory or whether protein dynamics would fall into clusters. Principal component analysis (PCA) of the molecular trajectories was performed, identifying 19 clusters by clustering in 6 dimensions (see FIG. 4 , where FIG. 4A shows the results of hierarchical clustering applied to the PCA subspaces from each trajectory, and FIG. 4B shows the overlap between subspaces for each pair of trajectories). As can be seen on FIG. 4B, the range in subspace overlap was 0.41-0.68. The high value of 0.68 indicates that the dynamics of these proteins follow similar motions. The trajectories represent a collection of possible folds. The inventors were interested in identifying folds which are stable and could be exploited for structure-based drug design purposes. For this purpose, they aimed to make a reasonable wide selection of possible Tau97 conformations to explore, which could be used as a starting point to identify transiently stable cryptic ligand-binding pockets of Tau97 which could be used for structure-based design, using LMT as a molecular probe (as explained in Example 2). The top 20 protein conformations closest in distance (i.e. the 20 closest neighbours) to the centroid of each cluster (i.e. a total of 380 protein conformations) were selected to give a representation of conformational space around the centroid. The root mean square deviation (RMSD) between these conformations was calculated (see FIG. 5 ) and used to select a diverse set of 178 conformations, for evaluation with molecular docking software.

Example 2—Analysis of the Structure of LMT-Bound dGAE

Overview

Molecular docking was performed using LMT to identify stable LMT-bound conformations of dGAE. As above, a slightly extended version of dGAE (Tau97) was used. However, the results herein are believed to be unaffected by the presence of 2 additional N-terminal residues, and equally apply to dGAE. As such, any references to “Tau97” in this example apply equally to dGAE.

Methods

Ligand parametrisation: Optimised structures and electrostatic potentials for the ligands were calculated at the MP2/6-311 G* level using General Atomic and Molecular Structure System (GAMESS) (Schmidt et al., 1993; Guest et al., 2005). Non-standard amino acid residues and ligands not described in the parameterisation libraries in LEaP (Schafmeister et al., 1995) were parameterised using the AMBER utility antechamber (Wang et al., 2006) and general AMBER force field (GAFF) (Wang et al., 2004). Ligand parameterisations were checked against the QM calculations by in vacuo MD simulations with sander (Crowley et al., 1997; Pearlman et al., 1995).

Docking and scoring (FIG. 3 , Step 330): Molecular docking analysis was performed using the MOE software from CCG. Ligand placement was determined using the Triangular Placement method and 10 poses were trained per query. A total of 65 ligand binding modes were identified with a docking score of <−8.5.

Iterative MD sampling of LMT bound conformations (FIG. 3 , Steps 340-350): To assess the strength of the binding interactions identified, the protein ligand complexes were subject to 1 ns of molecular dynamics simulations. The systems were equilibrated as described previously and a 1 ns production run was performed using the AMBER software package. The RMSD of the ligands from their initial binding poses were measured and docking scores re-measured for ligands which remained bound to the protein. Further rounds of 10 ns production runs were performed to identify the most tightly bound ligands, and simulations where the ligand dissociated from the protein were not analysed further. After 40 ns, PCA analysis (FIG. 3 , Step 360) using PCAZip was performed on the remaining complexes to assess which complexes were exhibiting similar dynamics. Additionally, PCA analysis was used to analyse and compare the final 25,000 frames from each of these remaining simulation trajectories complexes to those of the final 25,000 frames of the 30 Tau97 simulations of Example 1. After 100 ns a single stable protein conformation was identified which showed consistent tight ligand association and small protein residue RMSD fluctuations (i.e. below about 2.5 Å) as determined by PCA analysis. Protein-ligand interactions and intramolecular interactions within the uniquely identified protein-ligand complex were compared to the 30 Tau97 simulations of Example 1 (final 10 ns of the 50 ns simulations).

Results

In particular, protein docking was performed using LMT against the 178 protein conformations identified in Example 1. This generated 15,090 ligand poses which were refined based on the placement scoring function, with a cutoff of −80 kcal/mol) as determined by visual inspection to 65 candidate binding modes with a docking score <−8.5. The docking scores are based on the assumption that the protein is in a stable conformation, which may not be the case. Therefore, the protein ligand complexes were minimised in explicit solvent and then a 1 ns molecular dynamics run was performed. Complexes that were observed to dissociate from the complex during the 1 ns simulation (based on the RMSD of the ligand during the minimization as well as the ligand binding energy as determined from the docking scoring function) were excluded. This excluded 21 complexes where the ligand was weakly bound. The remaining complexes were subject to a further 10 ns molecular dynamics simulation. Complexes where the LMT ligands had dissociated from Tau97 at that point were eliminated (16 complexes). Further rounds of 10 ns simulation were performed, followed by calculation of the RMSD and ligand binding energy and elimination of the complexes where the protein was too flexible and/or the ligand not tightly bound as determined by visual inspection of the trajectories.

After 40 ns (i.e. a total of 50 ns), 22 complexes remained. PCA analysis of these 22 complexes was performed to assess which complexes were exhibiting similar dynamics. FIG. 6 shows the results of this analysis (pair-wise PCA subspace overlap), with subspaces overlaps ranging between 0.38 and 0.72. An examination of this subspace overlap clearly identified protein complex trajectories which were similar. For example, the range of subspace overlap for complexes 9, 10 and 11 was 0.68 to 0.72, and for complexes 15-18 the subspace overlap was between 0.57 to 0.69. Clustering of these trajectories helped to reduce the number of protein complexes used for further analysis. Comparison of the complexed structures to the trajectory of the 30 Tau97 structures from Example 1 was performed using PCA in an effort to identify unique protein dynamics induced by ligand binding. The results of this analysis are shown on FIG. 7 , which shows the PCA Subspace overlap comparison between the Tau97 conformations from Example 1 and the 22 ligand-bound conformations identified through molecular docking and MD sampling. The inventors were interested in whether there was conformational funnelling whereby a large set of different starting protein conformations converged to a smaller set of more similar conformations upon ligand binding, or whether ligand binding was non-specific. Here the range in subspace overlap was 0.34-0.61. The lower value of 0.34 in contrast to the Tau97 subspace overlap comparison (FIG. 4B, range 0.41-0.68) shows that ligand binding allowed the protein to explore new conformation space.

Simulations were continued for a total of 100 ns. A single complex was identified where the ligand remained tightly bound and the protein structure did not vary greatly after a 100 ns of molecular dynamics simulation time, with an average RMSD between frames varying by about 0.5±0.1 Å on average (as shown on FIG. 8 ). The three-dimensional coordinates of the protein conformation in this complex are provided in Table 1 and further representations of the complex are shown on FIG. 9 . This complex corresponds to LMT-bound conformation number 3 in FIGS. 6 and 7 . As can be seen on FIG. 7 , the protein fold induced upon LMT binding in complex 3 is relatively close to some of the conformations explored with the Tau97 protein as determined by PCA subspace overlap comparison of the molecular dynamics trajectories (PCA subspace overlap ranging between 0.37 and 0.53). Protein-ligand interactions and intramolecular interactions within the uniquely identified protein-ligand complex (LMT-bound conformation number 3) were compared to the 30 Tau97 conformations from the simulations of Example 1 (final 10 ns of the 50 ns simulations), and the results of this are shown on FIG. 10 . FIG. 10 shows the distance between pairs of heavy atoms as indicated, in the final 10 ns of the 50 ns simulations of Example 1 (for each of the 30 simulations) and in the final 10 ns of the 100 ns simulation for LMT-bound complex 3 (extreme left on the x axis).

The structure of complex 3 is stabilized by a number of strong hydrogen bonds (see FIG. 11 which shows close-up views of some of these bonds). The Glu342 carboxylic acid makes hydrogen bonds to the backbone NH of Val318 and the sidechain OH of Thr319 (see FIG. 11 , bottom left). Each amino acid along the sequence Gly367 to Lys375 form a number of important stabilising hydrogen bonds. Residues Lys369-Thr377 are involved in multiple hydrogen bonds to a tight hairpin formed by residue Ser341-Gln351 (see FIG. 11 , top left). Residue Gln351 forms hydrogen bonds through the backbone carbonyl to the Thr373 hydroxyl sidechain (see FIG. 11 , top left; distance between Gln351 O and Thr373 OH during the final 10 ns of a 50 ns simulation=2-4 Å, calculated as RMSD between frames of the final 10 ns of the simulation—see FIG. 10A which also shows that the Tau97 conformation 3 of Example 1 has a similar interaction). Residue Gln351 also forms hydrogen bonds through the sidechain carbonyl to the His374 backbone NH (see FIG. 11 , top left; distance between Gln351 C═O and His374 NH during the final 10 ns of a 50 ns simulation=2-4 Å—see FIG. 10B which also shows that the dGAE97 conformation 3 of Example 1 has a similar interaction) and to the Lys375 backbone NH (see FIG. 11 , top left; distance between Gln351 C═O and Lys375 NH during the final 10 ns of a 50 ns simulation=2.5-5 Å—see FIG. 10C which also shows that the Tau97 conformation 3 of Example 1 has a similar interaction). Arg349 forms three hydrogen bonds to Thr377 (distance between Arg349 O and Thr377 OH during the final 10 ns of a 50 ns simulation=2.5-4 Å, see FIG. 10D). The positively charged sidechain of Arg349 makes hydrogen bonds to the Thr377 hydroxyl sidechain and backbone, and the carbonyl backbone of Arg349 forms a hydrogen bond to the hydroxyl sidechain of Thr377. The carboxylic acid side chain of Glu372 forms multiple hydrogen bonding interactions with Ser356 (see FIG. 11 , top middle; distance between Gln372 C═O and Ser356 NH during the final 10 ns of a 50 ns simulation=2-4; 4.5-10 Å, see FIG. 10F) and Lys369. The Ser316 backbone carbonyl makes a hydrogen bond to the backbone NH of Ile371, and the Asp358 sidechain carbonyl makes a hydrogen bond to the Lys370 sidechain amine (see FIG. 11 , top right). While some of these interactions are also present in non-LMT bound conformations identified in Example 1, the hydrogen bonding distances are maintained for longer durations for the LMT complex and show considerably less variation than in the conformations identified in example, indicating that LMT has a stabilising influence on these otherwise transient interactions.

The LMT-bound conformation is a compact folded state with no beta sheets (or at least no beta sheets that are intrinsic to the stability of the LMT-bound complex). In other words, the LMT-bound conformation is a compact folded state where any beta sheets that may be present would be in the N or C-termini (which are dynamic sections and could adopt beta sheet conformations at least transiently) and would not be intrinsic to the stability of the LMT-bound conformation of the Tau97/dGAE protein. Without wishing to be bound by theory, the present inventors believe that the absence of beta sheets in this conformation that contributes to inhibition of assembly of the protein into oligomers, such as e.g. PHF. This compact folded state is very different from the structured conformation in PHFs. The compact folded state shows a tight hair pin loop between residues Val337-Gly355. Residues Val363-Gly367 contain a PGGG sequence which is threaded between the PGGG sequence Pro332-Gly335 and a loop formed by the sequence Thr319-Lys331. Further, residues Lys369-Thr377 are sandwiched between residues Asp314-Ser316, with close distances (about 2.5-5.0 Å) between the Ser316 beta-carbon and Thr373 backbone carbonyl (see FIG. 10E, which also shows that this interaction is also observed in Tau97 conformations 3 and 13 from Example 1). This is also the case in some of the conformations identified in example 1, but again the variance and distance between these atoms is smaller for the LMT-bound complex, indicating that LMT stabilises these interactions.

Comparing the starting conformation in Example 1 (which is based on cryo-EM analysis of paired helical filaments-like structures) to the LMT-bound conformation identified herein reveals that residues Gly355-Gly367 and Asn368-Arg379 are brought together in close proximity in the LMT-bound conformation, from a distance of over 36 Å to within hydrogen bonding distance (see FIG. 10G and FIG. 11 , bottom right). This is facilitated by the tight loop Val363-PG-Gly366, which enables the sequence Asn368-Arg379 to become folded back towards Lys343-Ser352 (which form a tight loop sometimes referred to as “hairpin loop”). Glu338 is folded towards Val363 (distance between Glu338 C═O and Val363 NH during the final 10 ns of a 50 ns simulation=2-4 Å, see FIG. 10G), a feature which is seen in many of the 30 simulations in example 1, suggesting that without the cross-β sheet structure within PHFs, a monomeric Tau97 protein will favor a wrapped-up conformation. The folding of the protein from a relatively extended conformation in the PHF-like state (starting point of the simulations in Example 1, see FIG. 9A) to the stabilised LMT-bound conformation identified herein reduces the total water accessible surface area over 20%, from about 10223 Å² to about 8014 Å². This results in a reduction in both polar (22%) and hydrophobic (21%) surface areas, indicating that a driving force in forming the compact LMT-bound conformation is the formation on intra-molecular hydrogen bonds and the burying of lipophilic side chains.

TABLE 1 Three-dimensional coordinates of Tau97 conformation with binding pocket. In this table, occupancy = 1 and temp factor = 0 for all atoms. Atom Residue Residue c name name number X (Å) Y (Å) Z (Å) Element 1 N ASP 295 −11.922 −4.691 18.009 N1+ 2 CA ASP 295 −10.501 −5.028 17.787 C 3 CB ASP 295 −9.613 −3.786 17.97 C 4 CG ASP 295 −9.828 −2.75 16.863 C 5 OD1 ASP 295 −10.766 −2.921 16.049 O 6 OD2 ASP 295 −9.098 −1.733 16.852 O1− 7 C ASP 295 −10.06 −6.185 18.683 C 8 O ASP 295 −10.789 −6.605 19.584 O 9 H1 ASP 295 −12.075 −4.43 18.971 H 10 H2 ASP 295 −12.516 −5.483 17.792 H 11 H3 ASP 295 −12.198 −3.911 17.421 H 12 HA ASP 295 −10.38 −5.361 16.756 H 13 HB2 ASP 295 −8.563 −4.079 17.961 H 14 HB3 ASP 295 −9.83 −3.335 18.939 H 15 N ASN 296 −8.872 −6.715 18.399 N 16 CA ASN 296 −8.244 −7.877 19.021 C 17 CB ASN 296 −8.589 −9.112 18.176 C 18 CG ASN 296 −10.045 −9.523 18.222 C 19 OD1 ASN 296 −10.49 −10.152 19.17 O 20 ND2 ASN 296 −10.817 −9.209 17.212 N 21 C ASN 296 −6.711 −7.728 19.066 C 22 O ASN 296 −6.133 −6.91 18.35 O 23 H ASN 296 −8.36 −6.316 17.614 H 24 HA ASN 296 −8.61 −7.998 20.038 H 25 HB2 ASN 296 −8.311 −8.896 17.148 H 26 HB3 ASN 296 −8.007 −9.971 18.509 H 27 HD21 ASN 296 −10.492 −8.577 16.486 H 28 HD22 ASN 296 −11.793 −9.487 17.26 H 29 N ILE 297 −6.05 −8.63 19.797 N 30 CA ILE 297 −4.609 −8.912 19.696 C 31 CB ILE 297 −3.801 −8.109 20.748 C 32 CG2 ILE 297 −4.238 −8.425 22.191 C 33 CG1 ILE 297 −2.282 −8.342 20.574 C 34 CD1 ILE 297 −1.393 −7.351 21.331 C 35 C ILE 297 −4.366 −10.426 19.778 C 36 O ILE 297 −5.035 −11.127 20.546 O 37 H ILE 297 −6.591 −9.271 20.358 H 38 HA ILE 297 −4.274 −8.588 18.709 H 39 HB ILE 297 −3.999 −7.052 20.562 H 40 HG12 ILE 297 −2.023 −9.353 20.892 H 41 HG13 ILE 297 −2.032 −8.245 19.522 H 42 HG21 ILE 297 −5.313 −8.288 22.303 H 43 HG22 ILE 297 −3.983 −9.452 22.453 H 44 HG23 ILE 297 −3.745 −7.746 22.886 H 45 HD11 ILE 297 −1.634 −6.335 21.019 H 46 HD12 ILE 297 −1.533 −7.452 22.407 H 47 HD13 ILE 297 −0.348 −7.552 21.095 H 48 N LYS 298 −3.421 −10.939 18.975 N 49 CA LYS 298 −2.97 −12.348 18.974 C 50 CB LYS 298 −4.038 −13.236 18.298 C 51 CG LYS 298 −4.345 −12.894 16.83 C 52 CD LYS 298 −5.377 −13.893 16.29 C 53 CE LYS 298 −5.71 −13.638 14.818 C 54 NZ LYS 298 −6.599 −14.701 14.304 N1+ 55 C LYS 298 −1.591 −12.506 18.313 C 56 O LYS 298 −0.992 −11.523 17.879 O 57 H LYS 298 −2.95 −10.297 18.338 H 58 HA LYS 298 −2.87 −12.679 20.009 H 59 HB2 LYS 298 −4.965 −13.159 18.865 H 60 HB3 LYS 298 −3.719 −14.278 18.359 H 61 HG2 LYS 298 −3.432 −12.958 16.239 H 62 HG3 LYS 298 −4.748 −11.883 16.759 H 63 HD2 LYS 298 −6.294 −13.822 16.879 H 64 HD3 LYS 298 −4.974 −14.901 16.395 H 65 HE2 LYS 298 −4.786 −13.624 14.243 H 66 HE3 LYS 298 −6.188 −12.661 14.717 H 67 HZ1 LYS 298 −7.48 −14.699 14.825 H 68 HZ2 LYS 298 −6.843 −14.561 13.329 H 69 HZ3 LYS 298 −6.156 −15.611 14.406 H 70 N HIE 299 −1.08 −13.737 18.198 N 71 CA HIE 299 0.014 −14.061 17.266 C 72 CB HIE 299 1.001 −15.067 17.882 C 73 CG HIE 299 2.186 −15.33 16.979 C 74 ND1 HIE 299 2.181 −16.167 15.859 N 75 CE1 HIE 299 3.375 −16.001 15.266 C 76 NE2 HIE 299 4.126 −15.135 15.962 N 77 CD2 HIE 299 3.392 −14.695 17.043 C 78 C HIE 299 −0.544 −14.585 15.935 C 79 O HIE 299 −1.36 −15.506 15.926 O 80 H HIE 299 −1.622 −14.509 18.559 H 81 HA HIE 299 0.583 −13.157 17.051 H 82 HB2 HIE 299 1.364 −14.681 18.834 H 83 HB3 HIE 299 0.487 −16.011 18.068 H 84 HD2 HIE 299 3.687 −13.954 17.775 H 85 HE2 HIE 299 5.067 −14.841 15.698 H 86 HE1 HIE 299 3.69 −16.5 14.358 H 87 N VAL 300 −0.054 −14.036 14.821 N 88 CA VAL 300 −0.168 −14.589 13.457 C 89 CB VAL 300 −1.297 −13.939 12.627 C 90 CG1 VAL 300 −2.666 −14.318 13.184 C 91 CG2 VAL 300 −1.205 −12.411 12.56 C 92 C VAL 300 1.175 −14.414 12.733 C 93 O VAL 300 1.965 −13.567 13.161 O 94 H VAL 300 0.624 −13.296 14.925 H 95 HA VAL 300 −0.372 −15.656 13.539 H 96 HB VAL 300 −1.243 −14.323 11.609 H 97 HG11 VAL 300 −3.446 −13.951 12.516 H 98 HG12 VAL 300 −2.743 −15.402 13.257 H 99 HG13 VAL 300 −2.789 −13.887 14.175 H 100 HG21 VAL 300 −1.382 −11.989 13.546 H 101 HG22 VAL 300 −0.225 −12.101 12.197 H 102 HG23 VAL 300 −1.961 −12.03 11.873 H 103 N PRO 301 1.483 −15.161 11.659 N 104 CD PRO 301 0.732 −16.291 11.124 C 105 CG PRO 301 1.79 −17.243 10.575 C 106 CB PRO 301 2.843 −16.275 10.044 C 107 CA PRO 301 2.819 −15.133 11.063 C 108 C PRO 301 3.273 −13.793 10.449 C 109 O PRO 301 2.526 −12.811 10.318 O 110 HA PRO 301 3.528 −15.387 11.852 H 111 HB2 PRO 301 2.541 −15.906 9.065 H 112 HB3 PRO 301 3.824 −16.744 9.989 H 113 HG2 PRO 301 1.395 −17.887 9.791 H 114 HG3 PRO 301 2.209 −17.838 11.388 H 115 HD2 PRO 301 0.086 −15.948 10.314 H 116 HD3 PRO 301 0.143 −16.804 11.884 H 117 N GLY 302 4.561 −13.774 10.109 N 118 CA GLY 302 5.28 −12.647 9.524 C 119 C GLY 302 6.091 −11.828 10.529 C 120 O GLY 302 6.22 −12.193 11.698 O 121 H GLY 302 5.11 −14.59 10.351 H 122 HA2 GLY 302 4.582 −11.983 9.017 H 123 HA3 GLY 302 5.964 −13.039 8.778 H 124 N GLY 303 6.675 −10.745 10.029 N 125 CA GLY 303 7.471 −9.755 10.758 C 126 C GLY 303 8.18 −8.857 9.742 C 127 O GLY 303 8.794 −9.376 8.818 O 128 H GLY 303 6.561 −10.592 9.03 H 129 HA2 GLY 303 8.218 −10.248 11.38 H 130 HA3 GLY 303 6.814 −9.161 11.391 H 131 N GLY 304 7.949 −7.544 9.765 N 132 CA GLY 304 8.306 −6.621 8.669 C 133 C GLY 304 7.413 −6.724 7.414 C 134 O GLY 304 7.299 −5.754 6.665 O 135 H GLY 304 7.466 −7.169 10.575 H 136 HA2 GLY 304 9.334 −6.824 8.361 H 137 HA3 GLY 304 8.258 −5.598 9.039 H 138 N SER 305 6.7 −7.844 7.257 N 139 CA SER 305 5.646 −8.136 6.276 C 140 CB SER 305 6.319 −8.675 5.006 C 141 OG SER 305 5.416 −8.856 3.929 O 142 C SER 305 4.644 −9.155 6.87 C 143 O SER 305 4.712 −9.491 8.058 O 144 H SER 305 6.917 −8.598 7.894 H 145 HA SER 305 5.112 −7.226 6.018 H 146 HB2 SER 305 7.089 −7.971 4.694 H 147 HB3 SER 305 6.797 −9.626 5.228 H 148 HG SER 305 5.964 −9.167 3.169 H 149 N VAL 306 3.751 −9.711 6.041 N 150 CA VAL 306 3.028 −10.977 6.321 C 151 CB VAL 306 1.807 −11.164 5.396 C 152 CG1 VAL 306 0.849 −9.974 5.501 C 153 CG2 VAL 306 2.19 −11.372 3.924 C 154 C VAL 306 3.95 −12.204 6.224 C 155 O VAL 306 3.698 −13.229 6.857 O 156 H VAL 306 3.756 −9.366 5.088 H 157 HA VAL 306 2.653 −10.943 7.344 H 158 HB VAL 306 1.264 −12.049 5.726 H 159 HG11 VAL 306 1.307 −9.069 5.104 H 160 HG12 VAL 306 −0.053 −10.192 4.931 H 161 HG13 VAL 306 0.57 −9.818 6.544 H 162 HG21 VAL 306 2.828 −10.562 3.58 H 163 HG22 VAL 306 2.714 −12.32 3.801 H 164 HG23 VAL 306 1.291 −11.408 3.307 H 165 N GLN 307 5.058 −12.063 5.493 N 166 CA GLN 307 6.253 −12.908 5.556 C 167 CB GLN 307 6.932 −12.9 4.178 C 168 CG GLN 307 6.122 −13.615 3.091 C 169 CD GLN 307 6.786 −13.446 1.732 C 170 OE1 GLN 307 6.659 −12.404 1.1 O 171 NE2 GLN 307 7.592 −14.388 1.29 N 172 C GLN 307 7.222 −12.376 6.628 C 173 O GLN 307 7.053 −11.26 7.12 O 174 H GLN 307 5.164 −11.182 5.014 H 175 HA GLN 307 5.979 −13.932 5.815 H 176 HB2 GLN 307 7.104 −11.865 3.88 H 177 HB3 GLN 307 7.901 −13.385 4.249 H 178 HG2 GLN 307 6.04 −14.675 3.331 H 179 HG3 GLN 307 5.12 −13.192 3.039 H 180 HE21 GLN 307 7.997 −14.284 0.372 H 181 HE22 GLN 307 7.68 −15.272 1.769 H 182 N ILE 308 8.26 −13.141 6.976 N 183 CA ILE 308 9.336 −12.678 7.867 C 184 CB ILE 308 9.885 −13.833 8.736 C 185 CG2 ILE 308 10.974 −13.285 9.681 C 186 CG1 ILE 308 8.771 −14.518 9.566 C 187 CD1 ILE 308 9.216 −15.845 10.196 C 188 C ILE 308 10.426 −12.021 7.008 C 189 O ILE 308 11.096 −12.698 6.222 O 190 H ILE 308 8.382 −14.04 6.514 H 191 HA ILE 308 8.94 −11.923 8.547 H 192 HB ILE 308 10.331 −14.579 8.076 H 193 HG12 ILE 308 8.431 −13.842 10.352 H 194 HG13 ILE 308 7.918 −14.747 8.929 H 195 HG21 ILE 308 11.419 −14.094 10.258 H 196 HG22 ILE 308 11.776 −12.814 9.115 H 197 HG23 ILE 308 10.541 −12.551 10.362 H 198 HD11 ILE 308 9.545 −16.53 9.415 H 199 HD12 ILE 308 10.031 −15.692 10.901 H 200 HD13 ILE 308 8.378 −16.289 10.732 H 201 N VAL 309 10.609 −10.709 7.128 N 202 CA VAL 309 11.491 −9.885 6.283 C 203 CB VAL 309 10.658 −8.975 5.355 C 204 CG1 VAL 309 11.542 −8.213 4.363 C 205 CG2 VAL 309 9.658 −9.785 4.519 C 206 C VAL 309 12.416 −9.053 7.171 C 207 O VAL 309 11.981 −8.518 8.187 O 208 H VAL 309 10.035 −10.208 7.803 H 209 HA VAL 309 12.106 −10.53 5.657 H 210 HB VAL 309 10.103 −8.256 5.959 H 211 HG11 VAL 309 12.056 −8.903 3.694 H 212 HG12 VAL 309 10.917 −7.54 3.776 H 213 HG13 VAL 309 12.271 −7.597 4.883 H 214 HG21 VAL 309 8.94 −10.281 5.168 H 215 HG22 VAL 309 9.112 −9.116 3.855 H 216 HG23 VAL 309 10.183 −10.529 3.921 H 217 N TYR 310 13.701 −8.942 6.812 N 218 CA TYR 310 14.711 −8.344 7.698 C 219 CB TYR 310 16.129 −8.759 7.249 C 220 CG TYR 310 17.03 −7.695 6.632 C 221 CD1 TYR 310 17.792 −6.854 7.469 C 222 CE1 TYR 310 18.706 −5.93 6.926 C 223 CZ TYR 310 18.863 −5.846 5.527 C 224 OH TYR 310 19.719 −4.94 4.977 O 225 CE2 TYR 310 18.093 −6.678 4.685 C 226 CD2 TYR 310 17.18 −7.601 5.234 C 227 C TYR 310 14.542 −6.829 7.925 C 228 O TYR 310 15.056 −6.308 8.915 O 229 H TYR 310 14.016 −9.405 5.974 H 230 HA TYR 310 14.559 −8.793 8.681 H 231 HB2 TYR 310 16.637 −9.135 8.135 H 232 HB3 TYR 310 16.068 −9.608 6.566 H 233 HD1 TYR 310 17.669 −6.922 8.54 H 234 HD2 TYR 310 16.625 −8.261 4.581 H 235 HE1 TYR 310 19.279 −5.289 7.581 H 236 HE2 TYR 310 18.235 −6.631 3.616 H 237 HH TYR 310 20.251 −4.466 5.644 H 238 N LYS 311 13.767 −6.142 7.071 N 239 CA LYS 311 13.35 −4.734 7.197 C 240 CB LYS 311 14.229 −3.83 6.317 C 241 CG LYS 311 15.709 −3.835 6.737 C 242 CD LYS 311 16.626 −3.02 5.817 C 243 CE LYS 311 16.485 −3.482 4.362 C 244 NZ LYS 311 17.735 −3.291 3.606 N1+ 245 C LYS 311 11.874 −4.587 6.777 C 246 O LYS 311 11.412 −5.376 5.952 O 247 H LYS 311 13.319 −6.667 6.333 H 248 HA LYS 311 13.464 −4.43 8.236 H 249 HB2 LYS 311 14.136 −4.176 5.287 H 250 HB3 LYS 311 13.857 −2.805 6.37 H 251 HG2 LYS 311 15.798 −3.452 7.753 H 252 HG3 LYS 311 16.073 −4.858 6.726 H 253 HD2 LYS 311 16.389 −1.96 5.886 H 254 HD3 LYS 311 17.653 −3.16 6.159 H 255 HE2 LYS 311 16.228 −4.544 4.355 H 256 HE3 LYS 311 15.667 −2.93 3.891 H 257 HZ1 LYS 311 18.058 −2.324 3.612 H 258 HZ2 LYS 311 18.482 −3.844 4.004 H 259 HZ3 LYS 311 17.626 −3.611 2.646 H 260 N PRO 312 11.123 −3.594 7.288 N 261 CD PRO 312 11.476 −2.722 8.396 C 262 CG PRO 312 10.145 −2.324 9.023 C 263 CB PRO 312 9.231 −2.237 7.802 C 264 CA PRO 312 9.722 −3.384 6.915 C 265 C PRO 312 9.519 −3.079 5.421 C 266 O PRO 312 10.208 −2.224 4.857 O 267 HA PRO 312 9.173 −4.285 7.174 H 268 HB2 PRO 312 9.401 −1.283 7.3 H 269 HB3 PRO 312 8.18 −2.352 8.069 H 270 HG2 PRO 312 10.226 −1.377 9.555 H 271 HG3 PRO 312 9.799 −3.115 9.69 H 272 HD2 PRO 312 11.985 −1.84 8.009 H 273 HD3 PRO 312 12.09 −3.219 9.146 H 274 N VAL 313 8.547 −3.735 4.78 N 275 CA VAL 313 8.273 −3.592 3.331 C 276 CB VAL 313 7.469 −4.786 2.771 C 277 CG1 VAL 313 8.261 −6.087 2.936 C 278 CG2 VAL 313 6.08 −4.948 3.402 C 279 C VAL 313 7.614 −2.254 2.966 C 280 O VAL 313 7.025 −1.581 3.819 O 281 H VAL 313 8.033 −4.441 5.297 H 282 HA VAL 313 9.234 −3.603 2.815 H 283 HB VAL 313 7.322 −4.628 1.703 H 284 HG11 VAL 313 8.422 −6.307 3.992 H 285 HG12 VAL 313 7.72 −6.913 2.475 H 286 HG13 VAL 313 9.23 −5.987 2.448 H 287 HG21 VAL 313 6.166 −5.123 4.472 H 288 HG22 VAL 313 5.488 −4.051 3.227 H 289 HG23 VAL 313 5.57 −5.798 2.947 H 290 N ASP 314 7.714 −1.84 1.701 N 291 CA ASP 314 7.188 −0.56 1.197 C 292 CB ASP 314 8.191 0.002 0.174 C 293 CG ASP 314 7.829 1.369 −0.421 C 294 OD1 ASP 314 8.633 1.835 −1.262 O 295 OD2 ASP 314 6.795 1.977 −0.042 O1− 296 C ASP 314 5.784 −0.738 0.596 C 297 O ASP 314 5.635 −1.349 −0.464 O 298 H ASP 314 8.188 −2.441 1.029 H 299 HA ASP 314 7.118 0.159 2.015 H 300 HB2 ASP 314 9.162 0.093 0.664 H 301 HB3 ASP 314 8.301 −0.712 −0.644 H 302 N LEU 315 4.745 −0.22 1.265 N 303 CA LEU 315 3.345 −0.335 0.821 C 304 CB LEU 315 2.436 −0.692 2.01 C 305 CG LEU 315 2.766 −2.025 2.704 C 306 CD1 LEU 315 1.847 −2.188 3.914 C 307 CD2 LEU 315 2.586 −3.241 1.793 C 308 C LEU 315 2.843 0.896 0.046 C 309 O LEU 315 1.662 0.981 −0.285 O 310 H LEU 315 4.918 0.268 2.14 H 311 HA LEU 315 3.274 −1.153 0.104 H 312 HB2 LEU 315 2.492 0.114 2.741 H 313 HB3 LEU 315 1.407 −0.742 1.656 H 314 HG LEU 315 3.795 −2.011 3.061 H 315 HD11 LEU 315 2.014 −1.365 4.609 H 316 HD12 LEU 315 0.805 −2.178 3.602 H 317 HD13 LEU 315 2.053 −3.129 4.418 H 318 HD21 LEU 315 2.763 −4.16 2.352 H 319 HD22 LEU 315 1.579 −3.26 1.38 H 320 HD23 LEU 315 3.299 −3.198 0.971 H 321 N SER 316 3.739 1.806 −0.348 N 322 CA SER 316 3.428 2.94 −1.235 C 323 CB SER 316 4.459 4.05 −1.006 C 324 OG SER 316 5.71 3.792 −1.628 O 325 C SER 316 3.314 2.544 −2.724 C 326 O SER 316 3.472 3.388 −3.616 O 327 H SER 316 4.711 1.672 −0.083 H 328 HA SER 316 2.457 3.34 −0.938 H 329 HB2 SER 316 4.052 4.981 −1.4 H 330 HB3 SER 316 4.622 4.17 0.064 H 331 HG SER 316 6.201 3.133 −1.079 H 332 N LYS 317 3.176 1.237 −2.993 N 333 CA LYS 317 3.71 0.559 −4.179 C 334 CB LYS 317 5.204 0.311 −3.889 C 335 CG LYS 317 6.032 −0.045 −5.132 C 336 CD LYS 317 7.481 0.427 −4.973 C 337 CE LYS 317 8.244 −0.347 −3.895 C 338 NZ LYS 317 9.453 0.391 −3.471 N1+ 339 C LYS 317 2.975 −0.753 −4.497 C 340 O LYS 317 2.301 −1.328 −3.642 O 341 H LYS 317 2.897 0.644 −2.221 H 342 HA LYS 317 3.614 1.224 −5.039 H 343 HB2 LYS 317 5.617 1.226 −3.459 H 344 HB3 LYS 317 5.31 −0.477 −3.141 H 345 HG2 LYS 317 6.006 −1.122 −5.304 H 346 HG3 LYS 317 5.607 0.454 −6.003 H 347 HD2 LYS 317 7.996 0.304 −5.927 H 348 HD3 LYS 317 7.475 1.49 −4.727 H 349 HE2 LYS 317 7.602 −0.517 −3.026 H 350 HE3 LYS 317 8.525 −1.32 −4.307 H 351 HZ1 LYS 317 10.012 0.679 −4.272 H 352 HZ2 LYS 317 9.202 1.201 −2.897 H 353 HZ3 LYS 317 10.025 −0.214 −2.885 H 354 N VAL 318 3.174 −1.258 −5.716 N 355 CA VAL 318 2.666 −2.546 −6.217 C 356 CB VAL 318 1.349 −2.321 −6.999 C 357 CG1 VAL 318 1.558 −1.625 −8.351 C 358 CG2 VAL 318 0.553 −3.614 −7.215 C 359 C VAL 318 3.742 −3.265 −7.046 C 360 O VAL 318 4.704 −2.652 −7.511 O 361 H VAL 318 3.798 −0.759 −6.331 H 362 HA VAL 318 2.438 −3.184 −5.362 H 363 HB VAL 318 0.719 −1.67 −6.391 H 364 HG11 VAL 318 0.59 −1.402 −8.799 H 365 HG12 VAL 318 2.096 −0.688 −8.211 H 366 HG13 VAL 318 2.12 −2.269 −9.029 H 367 HG21 VAL 318 0.408 −4.12 −6.26 H 368 HG22 VAL 318 −0.426 −3.374 −7.63 H 369 HG23 VAL 318 1.064 −4.277 −7.911 H 370 N THR 319 3.611 −4.582 −7.19 N 371 CA THR 319 4.419 −5.435 −8.079 C 372 CB THR 319 4.325 −6.9 −7.611 C 373 CG2 THR 319 5.187 −7.88 −8.405 C 374 OG1 THR 319 4.766 −6.987 −6.269 O 375 C THR 319 3.975 −5.297 −9.543 C 376 O THR 319 2.808 −5.03 −9.831 O 377 H THR 319 2.861 −5.029 −6.675 H 378 HA THR 319 5.462 −5.127 −8.016 H 379 HB THR 319 3.286 −7.223 −7.652 H 380 HG21 THR 319 6.234 −7.578 −8.378 H 381 HG22 THR 319 5.077 −8.876 −7.98 H 382 HG23 THR 319 4.847 −7.941 −9.437 H 383 HG1 THR 319 4.001 −6.699 −5.718 H 384 N SER 320 4.9 −5.51 −10.486 N 385 CA SER 320 4.599 −5.542 −11.925 C 386 CB SER 320 5.892 −5.706 −12.726 C 387 OG SER 320 5.694 −5.251 −14.052 O 388 C SER 320 3.584 −6.631 −12.324 C 389 O SER 320 3.328 −7.587 −11.588 O 390 H SER 320 5.853 −5.678 −10.196 H 391 HA SER 320 4.163 −4.578 −12.188 H 392 HB2 SER 320 6.68 −5.106 −12.272 H 393 HB3 SER 320 6.197 −6.753 −12.725 H 394 HG SER 320 6.467 −5.545 −14.576 H 395 N LYS 321 3.003 −6.472 −13.517 N 396 CA LYS 321 1.883 −7.247 −14.079 C 397 CB LYS 321 0.614 −6.366 −14.015 C 398 CG LYS 321 0.143 −5.985 −12.59 C 399 CD LYS 321 −0.642 −4.659 −12.584 C 400 CE LYS 321 −1.207 −4.324 −11.194 C 401 NZ LYS 321 −1.814 −2.97 −11.171 N1+ 402 C LYS 321 2.241 −7.623 −15.526 C 403 O LYS 321 2.673 −6.744 −16.274 O 404 H LYS 321 3.378 −5.733 −14.098 H 405 HA LYS 321 1.722 −8.163 −13.505 H 406 HB2 LYS 321 0.818 −5.45 −14.574 H 407 HB3 LYS 321 −0.203 −6.885 −14.517 H 408 HG2 LYS 321 −0.483 −6.787 −12.198 H 409 HG3 LYS 321 0.995 −5.864 −11.923 H 410 HD2 LYS 321 0.029 −3.856 −12.897 H 411 HD3 LYS 321 −1.466 −4.724 −13.297 H 412 HE2 LYS 321 −1.968 −5.069 −10.946 H 413 HE3 LYS 321 −0.409 −4.387 −10.447 H 414 HZ1 LYS 321 −2.484 −2.845 −11.926 H 415 HZ2 LYS 321 −2.316 −2.766 −10.305 H 416 HZ3 LYS 321 −1.13 −2.226 −11.299 H 417 N CYS 322 2.154 −8.898 −15.91 N 418 CA CYS 322 2.797 −9.386 −17.141 C 419 CB CYS 322 2.809 −10.921 −17.134 C 420 SG CYS 322 3.917 −11.513 −15.822 S 421 C CYS 322 2.172 −8.829 −18.436 C 422 O CYS 322 0.957 −8.626 −18.521 O 423 H CYS 322 1.757 −9.581 −15.272 H 424 HA CYS 322 3.836 −9.05 −17.132 H 425 HB2 CYS 322 1.797 −11.295 −16.971 H 426 HB3 CYS 322 3.168 −11.293 −18.096 H 427 HG CYS 322 5.07 −11.282 −16.471 H 428 N GLY 323 3.004 −8.638 −19.465 N 429 CA GLY 323 2.571 −8.235 −20.805 C 430 C GLY 323 1.992 −6.82 −20.868 C 431 O GLY 323 2.55 −5.874 −20.306 O 432 H GLY 323 3.983 −8.855 −19.341 H 433 HA2 GLY 323 3.423 −8.278 −21.483 H 434 HA3 GLY 323 1.822 −8.945 −21.158 H 435 N SER 324 0.848 −6.673 −21.539 N 436 CA SER 324 0.166 −5.401 −21.834 C 437 CB SER 324 −0.845 −5.619 −22.973 C 438 OG SER 324 −1.617 −6.787 −22.757 O 439 C SER 324 −0.456 −4.69 −20.615 C 440 O SER 324 −1.302 −3.813 −20.775 O 441 H SER 324 0.404 −7.502 −21.912 H 442 HA SER 324 0.917 −4.714 −22.22 H 443 HB2 SER 324 −1.497 −4.751 −23.076 H 444 HB3 SER 324 −0.292 −5.742 −23.905 H 445 HG SER 324 −2.213 −6.901 −23.508 H 446 N LEU 325 −0.001 −5.018 −19.4 N 447 CA LEU 325 −0.416 −4.421 −18.127 C 448 CB LEU 325 −0.895 −5.547 −17.189 C 449 CG LEU 325 −2.054 −6.407 −17.738 C 450 CD1 LEU 325 −2.353 −7.544 −16.76 C 451 CD2 LEU 325 −3.337 −5.599 −17.944 C 452 C LEU 325 0.716 −3.573 −17.513 C 453 O LEU 325 0.544 −2.373 −17.284 O 454 H LEU 325 0.722 −5.725 −19.37 H 455 HA LEU 325 −1.253 −3.745 −18.3 H 456 HB2 LEU 325 −0.05 −6.207 −16.99 H 457 HB3 LEU 325 −1.207 −5.1 −16.244 H 458 HG LEU 325 −1.764 −6.855 −18.688 H 459 HD11 LEU 325 −2.669 −7.142 −15.797 H 460 HD12 LEU 325 −3.148 −8.174 −17.163 H 461 HD13 LEU 325 −1.461 −8.155 −16.624 H 462 HD21 LEU 325 −3.629 −5.109 −17.015 H 463 HD22 LEU 325 −3.187 −4.85 −18.721 H 464 HD23 LEU 325 −4.141 −6.262 −18.266 H 465 N GLY 326 1.928 −4.129 −17.382 N 466 CA GLY 326 3.14 −3.353 −17.074 C 467 C GLY 326 3.542 −2.377 −18.193 C 468 O GLY 326 4.117 −1.324 −17.918 O 469 H GLY 326 2.029 −5.131 −17.506 H 470 HA2 GLY 326 2.983 −2.784 −16.157 H 471 HA3 GLY 326 3.965 −4.047 −16.915 H 472 N ASN 327 3.117 −2.655 −19.431 N 473 CA ASN 327 3.278 −1.797 −20.612 C 474 CB ASN 327 2.943 −2.678 −21.842 C 475 CG ASN 327 3.643 −2.325 −23.151 C 476 OD1 ASN 327 4.077 −3.195 −23.896 O 477 ND2 ASN 327 3.686 −1.072 −23.527 N 478 C ASN 327 2.422 −0.497 −20.557 C 479 O ASN 327 2.556 0.356 −21.433 O 480 H ASN 327 2.67 −3.552 −19.57 H 481 HA ASN 327 4.326 −1.493 −20.669 H 482 HB2 ASN 327 3.222 −3.71 −21.633 H 483 HB3 ASN 327 1.868 −2.646 −22.014 H 484 HD21 ASN 327 3.285 −0.355 −22.932 H 485 HD22 ASN 327 4.027 −0.855 −24.449 H 486 N ILE 328 1.526 −0.346 −19.571 N 487 CA ILE 328 0.665 0.835 −19.377 C 488 CB ILE 328 −0.684 0.411 −18.741 C 489 CG2 ILE 328 −1.583 1.638 −18.553 C 490 CG1 ILE 328 −1.434 −0.67 −19.551 C 491 CD1 ILE 328 −2.611 −1.297 −18.785 C 492 C ILE 328 1.402 1.874 −18.508 C 493 O ILE 328 1.855 1.535 −17.412 O 494 H ILE 328 1.47 −1.072 −18.869 H 495 HA ILE 328 0.455 1.283 −20.348 H 496 HB ILE 328 −0.477 0.001 −17.755 H 497 HG12 ILE 328 −1.796 −0.243 −20.487 H 498 HG13 ILE 328 −0.748 −1.478 −19.794 H 499 HG21 ILE 328 −1.065 2.378 −17.953 H 500 HG22 ILE 328 −1.844 2.064 −19.522 H 501 HG23 ILE 328 −2.495 1.373 −18.019 H 502 HD11 ILE 328 −3.401 −0.57 −18.616 H 503 HD12 ILE 328 −3.023 −2.124 −19.362 H 504 HD13 ILE 328 −2.267 −1.678 −17.823 H 505 N HIE 329 1.47 3.14 −18.941 N 506 CA HIE 329 2.339 4.176 −18.346 C 507 CB HIE 329 3.167 4.847 −19.451 C 508 CG HIE 329 3.957 3.888 −20.302 C 509 ND1 HIE 329 3.775 3.676 −21.674 N 510 CE1 HIE 329 4.707 2.771 −22.024 C 511 NE2 HIE 329 5.458 2.435 −20.961 N 512 CD2 HIE 329 4.995 3.121 −19.864 C 513 C HIE 329 1.595 5.26 −17.552 C 514 O HIE 329 0.539 5.713 −17.989 O 515 H HIE 329 1.028 3.36 −19.828 H 516 HA HIE 329 3.035 3.69 −17.669 H 517 HB2 HIE 329 2.502 5.42 −20.098 H 518 HB3 HIE 329 3.865 5.546 −18.989 H 519 HD2 HIE 329 5.39 3.088 −18.856 H 520 HE2 HIE 329 6.285 1.841 −20.987 H 521 HE1 HIE 329 4.856 2.388 −23.027 H 522 N HID 330 2.184 5.773 −16.462 N 523 CA HID 330 1.728 6.991 −15.757 C 524 CB HID 330 1.314 6.688 −14.301 C 525 CG HID 330 2.242 5.829 −13.472 C 526 ND1 HID 330 1.843 4.969 −12.472 N 527 CE1 HID 330 2.927 4.338 −11.997 C 528 NE2 HID 330 4.03 4.769 −12.631 N 529 CD2 HID 330 3.607 5.733 −13.556 C 530 C HID 330 2.701 8.186 −15.889 C 531 O HID 330 3.891 8.021 −16.183 O 532 H HID 330 3.054 5.349 −16.156 H 533 HA HID 330 0.814 7.333 −16.245 H 534 HB2 HID 330 1.15 7.627 −13.771 H 535 HB3 HID 330 0.35 6.179 −14.338 H 536 HD1 HID 330 0.903 4.879 −12.09 H 537 HD2 HID 330 4.245 6.278 −14.237 H 538 HE1 HID 330 2.917 3.605 −11.199 H 539 N LYS 331 2.166 9.412 −15.742 N 540 CA LYS 331 2.791 10.652 −16.235 C 541 CB LYS 331 2.088 11.007 −17.559 C 542 CG LYS 331 2.808 12.105 −18.359 C 543 CD LYS 331 2.059 12.467 −19.655 C 544 CE LYS 331 1.763 11.275 −20.581 C 545 NZ LYS 331 2.983 10.694 −21.186 N1+ 546 C LYS 331 2.716 11.811 −15.217 C 547 O LYS 331 1.623 12.313 −14.954 O 548 H LYS 331 1.193 9.474 −15.467 H 549 HA LYS 331 3.836 10.457 −16.473 H 550 HB2 LYS 331 2.053 10.104 −18.169 H 551 HB3 LYS 331 1.061 11.319 −17.356 H 552 HG2 LYS 331 2.891 13.005 −17.748 H 553 HG3 LYS 331 3.815 11.77 −18.605 H 554 HD2 LYS 331 1.105 12.919 −19.38 H 555 HD3 LYS 331 2.632 13.219 −20.2 H 556 HE2 LYS 331 1.224 10.508 −20.021 H 557 HE3 LYS 331 1.104 11.614 −21.384 H 558 HZ1 LYS 331 2.728 9.898 −21.772 H 559 HZ2 LYS 331 3.435 11.378 −21.786 H 560 HZ3 LYS 331 3.654 10.384 −20.488 H 561 N PRO 332 3.851 12.296 −14.681 N 562 CD PRO 332 5.141 11.626 −14.65 C 563 CG PRO 332 5.794 12.09 −13.353 C 564 CB PRO 332 5.308 13.534 −13.244 C 565 CA PRO 332 3.887 13.481 −13.819 C 566 C PRO 332 3.499 14.775 −14.558 C 567 O PRO 332 4.041 15.088 −15.623 O 568 HA PRO 332 3.193 13.321 −12.996 H 569 HB2 PRO 332 5.948 14.159 −13.865 H 570 HB3 PRO 332 5.315 13.887 −12.212 H 571 HG2 PRO 332 6.879 12.031 −13.415 H 572 HG3 PRO 332 5.415 11.504 −12.514 H 573 HD2 PRO 332 5.74 11.945 −15.505 H 574 HD3 PRO 332 5.039 10.54 −14.641 H 575 N GLY 333 2.598 15.569 −13.979 N 576 CA GLY 333 2.113 16.819 −14.57 C 577 C GLY 333 0.866 17.366 −13.873 C 578 O GLY 333 0.479 16.88 −12.815 O 579 H GLY 333 2.229 15.314 −13.065 H 580 HA2 GLY 333 2.897 17.574 −14.517 H 581 HA3 GLY 333 1.872 16.653 −15.621 H 582 N GLY 334 0.222 18.351 −14.502 N 583 CA GLY 334 −1.034 18.96 −14.054 C 584 C GLY 334 −2.087 19.023 −15.167 C 585 O GLY 334 −2.053 18.261 −16.137 O 586 H GLY 334 0.57 18.633 −15.414 H 587 HA2 GLY 334 −1.458 18.397 −13.221 H 588 HA3 GLY 334 −0.829 19.972 −13.705 H 589 N GLY 335 −3.039 19.944 −15.058 N 590 CA GLY 335 −4.211 20.012 −15.931 C 591 C GLY 335 −5.226 18.944 −15.539 C 592 O GLY 335 −5.713 18.954 −14.407 O 593 H GLY 335 −3.062 20.517 −14.22 H 594 HA2 GLY 335 −4.688 20.985 −15.821 H 595 HA3 GLY 335 −3.92 19.884 −16.973 H 596 N GLN 336 −5.54 18.023 −16.451 N 597 CA GLN 336 −6.463 16.915 −16.181 C 598 CB GLN 336 −6.677 16.059 −17.441 C 599 CG GLN 336 −7.199 16.838 −18.661 C 600 CD GLN 336 −6.099 17.578 −19.42 C 601 OE1 GLN 336 −5.088 17.006 −19.817 O 602 NE2 GLN 336 −6.222 18.869 −19.621 N 603 C GLN 336 −5.948 16.042 −15.022 C 604 O GLN 336 −4.773 15.678 −15.006 O 605 H GLN 336 −5.092 18.063 −17.353 H 606 HA GLN 336 −7.423 17.34 −15.888 H 607 HB2 GLN 336 −5.746 15.553 −17.703 H 608 HB3 GLN 336 −7.412 15.293 −17.195 H 609 HG2 GLN 336 −7.657 16.132 −19.354 H 610 HG3 GLN 336 −7.977 17.533 −18.343 H 611 HE21 GLN 336 −7.037 19.373 −19.267 H 612 HE22 GLN 336 −5.552 19.312 −20.241 H 613 N VAL 337 −6.817 15.683 −14.075 N 614 CA VAL 337 −6.434 15.028 −12.806 C 615 CB VAL 337 −7.162 15.7 −11.622 C 616 CG1 VAL 337 −6.864 15.029 −10.275 C 617 CG2 VAL 337 −6.737 17.171 −11.508 C 618 C VAL 337 −6.684 13.518 −12.851 C 619 O VAL 337 −7.755 13.071 −13.262 O 620 H VAL 337 −7.786 15.961 −14.193 H 621 HA VAL 337 −5.364 15.172 −12.647 H 622 HB VAL 337 −8.239 15.66 −11.792 H 623 HG11 VAL 337 −7.25 14.009 −10.268 H 624 HG12 VAL 337 −5.789 15.012 −10.091 H 625 HG13 VAL 337 −7.359 15.577 −9.473 H 626 HG21 VAL 337 −5.652 17.241 −11.425 H 627 HG22 VAL 337 −7.068 17.726 −12.385 H 628 HG23 VAL 337 −7.191 17.626 −10.628 H 629 N GLU 338 −5.708 12.713 −12.421 N 630 CA GLU 338 −5.815 11.248 −12.427 C 631 CB GLU 338 −4.402 10.639 −12.536 C 632 CG GLU 338 −4.35 9.143 −12.891 C 633 CD GLU 338 −5.285 8.774 −14.049 C 634 OE1 GLU 338 −6.426 8.347 −13.747 O 635 OE2 GLU 338 −4.956 8.958 −15.242 O1− 636 C GLU 338 −6.68 10.706 −11.261 C 637 O GLU 338 −6.755 11.302 −10.184 O 638 H GLU 338 −4.846 13.126 −12.09 H 639 HA GLU 338 −6.336 10.98 −13.342 H 640 HB2 GLU 338 −3.86 11.177 −13.316 H 641 HB3 GLU 338 −3.869 10.798 −11.597 H 642 HG2 GLU 338 −3.322 8.873 −13.14 H 643 HG3 GLU 338 −4.637 8.569 −12.007 H 644 N VAL 339 −7.378 9.589 −11.499 N 645 CA VAL 339 −8.361 8.936 −10.605 C 646 CB VAL 339 −9.808 9.303 −11.025 C 647 CG1 VAL 339 −10.828 8.958 −9.931 C 648 CG2 VAL 339 −10.014 10.789 −11.365 C 649 C VAL 339 −8.205 7.399 −10.601 C 650 O VAL 339 −8.596 6.732 −9.634 O 651 H VAL 339 −7.241 9.169 −12.414 H 652 HA VAL 339 −8.197 9.286 −9.585 H 653 HB VAL 339 −10.056 8.732 −11.918 H 654 HG11 VAL 339 −10.847 7.884 −9.749 H 655 HG12 VAL 339 −10.572 9.478 −9.007 H 656 HG13 VAL 339 −11.828 9.258 −10.243 H 657 HG21 VAL 339 −9.745 11.412 −10.513 H 658 HG22 VAL 339 −9.408 11.074 −12.224 H 659 HG23 VAL 339 −11.058 10.969 −11.622 H 660 N LYS 340 −7.584 6.817 −11.639 N 661 CA LYS 340 −7.233 5.39 −11.699 C 662 CB LYS 340 −6.742 5.002 −13.101 C 663 CG LYS 340 −7.901 5.036 −14.108 C 664 CD LYS 340 −7.462 4.716 −15.544 C 665 CE LYS 340 −6.448 5.709 −16.126 C 666 NZ LYS 340 −6.929 7.108 −16.076 N1+ 667 C LYS 340 −6.178 5.071 −10.645 C 668 O LYS 340 −5.176 5.768 −10.521 O 669 H LYS 340 −7.228 7.412 −12.384 H 670 HA LYS 340 −8.12 4.799 −11.476 H 671 HB2 LYS 340 −5.943 5.679 −13.406 H 672 HB3 LYS 340 −6.342 3.988 −13.074 H 673 HG2 LYS 340 −8.648 4.3 −13.806 H 674 HG3 LYS 340 −8.373 6.017 −14.083 H 675 HD2 LYS 340 −7.025 3.716 −15.563 H 676 HD3 LYS 340 −8.347 4.703 −16.183 H 677 HE2 LYS 340 −5.502 5.629 −15.581 H 678 HE3 LYS 340 −6.249 5.435 −17.163 H 679 HZ1 LYS 340 −6.988 7.425 −15.109 H 680 HZ2 LYS 340 −6.235 7.741 −16.465 H 681 HZ3 LYS 340 −7.846 7.243 −16.489 H 682 N SER 341 −6.433 4.039 −9.854 N 683 CA SER 341 −5.681 3.689 −8.648 C 684 CB SER 341 −6.241 4.451 −7.436 C 685 OG SER 341 −7.587 4.083 −7.197 O 686 C SER 341 −5.745 2.183 −8.384 C 687 O SER 341 −6.702 1.508 −8.766 O 688 H SER 341 −7.291 3.532 −10.014 H 689 HA SER 341 −4.639 3.98 −8.768 H 690 HB2 SER 341 −5.64 4.219 −6.556 H 691 HB3 SER 341 −6.19 5.525 −7.62 H 692 HG SER 341 −7.743 4.101 −6.234 H 693 N GLU 342 −4.774 1.669 −7.636 N 694 CA GLU 342 −4.743 0.277 −7.177 C 695 CB GLU 342 −3.264 −0.087 −6.945 C 696 CG GLU 342 −2.957 −1.587 −6.936 C 697 CD GLU 342 −3.262 −2.278 −8.273 C 698 OE1 GLU 342 −3.393 −3.521 −8.288 O 699 OE2 GLU 342 −3.378 −1.613 −9.33 O1− 700 C GLU 342 −5.625 0.045 −5.927 C 701 O GLU 342 −5.78 −1.092 −5.466 O 702 H GLU 342 −3.959 2.244 −7.423 H 703 HA GLU 342 −5.142 −0.352 −7.974 H 704 HB2 GLU 342 −2.657 0.357 −7.735 H 705 HB3 GLU 342 −2.939 0.348 −6 H 706 HG2 GLU 342 −1.898 −1.712 −6.71 H 707 HG3 GLU 342 −3.518 −2.065 −6.133 H 708 N LYS 343 −6.18 1.121 −5.346 N 709 CA LYS 343 −7.065 1.127 −4.17 C 710 CB LYS 343 −6.257 1.302 −2.867 C 711 CG LYS 343 −5.54 0.018 −2.429 C 712 CD LYS 343 −4.065 −0.084 −2.845 C 713 CE LYS 343 −3.477 −1.487 −2.625 C 714 NZ LYS 343 −4.315 −2.552 −3.234 N1+ 715 C LYS 343 −8.132 2.222 −4.254 C 716 O LYS 343 −7.818 3.373 −4.567 O 717 H LYS 343 −6.012 2.009 −5.798 H 718 HA LYS 343 −7.596 0.177 −4.12 H 719 HB2 LYS 343 −5.553 2.133 −2.949 H 720 HB3 LYS 343 −6.968 1.548 −2.079 H 721 HG2 LYS 343 −5.578 −0.049 −1.348 H 722 HG3 LYS 343 −6.107 −0.823 −2.819 H 723 HD2 LYS 343 −3.955 0.181 −3.892 H 724 HD3 LYS 343 −3.486 0.635 −2.262 H 725 HE2 LYS 343 −2.478 −1.511 −3.073 H 726 HE3 LYS 343 −3.365 −1.669 −1.552 H 727 HZ1 LYS 343 −3.863 −3.465 −3.174 H 728 HZ2 LYS 343 −5.198 −2.636 −2.739 H 729 HZ3 LYS 343 −4.502 −2.359 −4.212 H 730 N LEU 344 −9.362 1.863 −3.879 N 731 CA LEU 344 −10.538 2.735 −3.707 C 732 CB LEU 344 −11.49 2.547 −4.907 C 733 CG LEU 344 −11.01 3.106 −6.259 C 734 CD1 LEU 344 −12.063 2.805 −7.326 C 735 CD2 LEU 344 −10.807 4.621 −6.208 C 736 C LEU 344 −11.306 2.455 −2.394 C 737 O LEU 344 −11.918 3.361 −1.826 O 738 H LEU 344 −9.505 0.878 −3.701 H 739 HA LEU 344 −10.216 3.775 −3.658 H 740 HB2 LEU 344 −11.703 1.483 −5.019 H 741 HB3 LEU 344 −12.434 3.039 −4.671 H 742 HG LEU 344 −10.074 2.625 −6.546 H 743 HD11 LEU 344 −12.215 1.729 −7.403 H 744 HD12 LEU 344 −13.008 3.279 −7.063 H 745 HD13 LEU 344 −11.734 3.181 −8.294 H 746 HD21 LEU 344 −11.704 5.1 −5.816 H 747 HD22 LEU 344 −9.953 4.864 −5.577 H 748 HD23 LEU 344 −10.605 5.006 −7.206 H 749 N ASP 345 −11.217 1.234 −1.861 N 750 CA ASP 345 −11.762 0.816 −0.563 C 751 CB ASP 345 −12.233 −0.646 −0.637 C 752 CG ASP 345 −13.553 −0.8 −1.383 C 753 OD1 ASP 345 −13.532 −0.95 −2.629 O 754 OD2 ASP 345 −14.61 −0.727 −0.708 O1− 755 C ASP 345 −10.715 0.96 0.55 C 756 O ASP 345 −9.581 0.501 0.385 O 757 H ASP 345 −10.748 0.523 −2.404 H 758 HA ASP 345 −12.619 1.442 −0.312 H 759 HB2 ASP 345 −11.465 −1.259 −1.111 H 760 HB3 ASP 345 −12.373 −1.019 0.38 H 761 N PHE 346 −11.111 1.521 1.696 N 762 CA PHE 346 −10.254 1.734 2.875 C 763 CB PHE 346 −10.987 2.655 3.865 C 764 CG PHE 346 −11.454 3.988 3.306 C 765 CD1 PHE 346 −12.829 4.29 3.246 C 766 CE1 PHE 346 −13.258 5.551 2.796 C 767 CZ PHE 346 −12.314 6.513 2.393 C 768 CE2 PHE 346 −10.943 6.211 2.437 C 769 CD2 PHE 346 −10.512 4.954 2.9 C 770 C PHE 346 −9.833 0.42 3.571 C 771 O PHE 346 −10.364 −0.653 3.27 O 772 H PHE 346 −12.082 1.793 1.77 H 773 HA PHE 346 −9.34 2.238 2.557 H 774 HB2 PHE 346 −11.845 2.116 4.268 H 775 HB3 PHE 346 −10.322 2.869 4.702 H 776 HD1 PHE 346 −13.564 3.569 3.577 H 777 HD2 PHE 346 −9.454 4.743 2.973 H 778 HE1 PHE 346 −14.315 5.784 2.784 H 779 HE2 PHE 346 −10.218 6.958 2.142 H 780 HZ PHE 346 −12.64 7.49 2.06 H 781 N LYS 347 −8.887 0.493 4.52 N 782 CA LYS 347 −8.403 −0.654 5.318 C 783 CB LYS 347 −7.213 −1.295 4.564 C 784 CG LYS 347 −6.626 −2.5 5.307 C 785 CD LYS 347 −5.581 −3.31 4.541 C 786 CE LYS 347 −4.922 −4.355 5.451 C 787 NZ LYS 347 −4.123 −3.751 6.546 N1+ 788 C LYS 347 −8.091 −0.268 6.78 C 789 O LYS 347 −7.999 0.914 7.103 O 790 H LYS 347 −8.502 1.408 4.734 H 791 HA LYS 347 −9.2 −1.399 5.368 H 792 HB2 LYS 347 −7.558 −1.624 3.582 H 793 HB3 LYS 347 −6.429 −0.549 4.423 H 794 HG2 LYS 347 −6.139 −2.11 6.185 H 795 HG3 LYS 347 −7.429 −3.165 5.615 H 796 HD2 LYS 347 −6.069 −3.818 3.708 H 797 HD3 LYS 347 −4.817 −2.644 4.155 H 798 HE2 LYS 347 −5.708 −4.98 5.882 H 799 HE3 LYS 347 −4.284 −4.998 4.836 H 800 HZ1 LYS 347 −4.7 −3.171 7.154 H 801 HZ2 LYS 347 −3.738 −4.483 7.146 H 802 HZ3 LYS 347 −3.361 −3.2 6.184 H 803 N ASP 348 −8.012 −1.257 7.675 N 804 CA ASP 348 −7.563 −1.149 9.075 C 805 CB ASP 348 −8.406 −2.123 9.916 C 806 CG ASP 348 −8.022 −2.085 11.391 C 807 OD1 ASP 348 −7.458 −3.089 11.868 O 808 OD2 ASP 348 −8.12 −1.014 12.031 O1− 809 C ASP 348 −6.036 −1.389 9.263 C 810 O ASP 348 −5.369 −1.997 8.417 O 811 H ASP 348 −8.244 −2.187 7.348 H 812 HA ASP 348 −7.78 −0.14 9.432 H 813 HB2 ASP 348 −9.462 −1.878 9.819 H 814 HB3 ASP 348 −8.26 −3.135 9.534 H 815 N ARG 349 −5.485 −0.934 10.402 N 816 CA ARG 349 −4.047 −0.852 10.77 C 817 CB ARG 349 −3.384 −2.236 10.953 C 818 CG ARG 349 −4 −3.22 11.961 C 819 CD ARG 349 −4.173 −2.685 13.389 C 820 NE ARG 349 −5.513 −2.116 13.602 N 821 CZ ARG 349 −6.101 −1.835 14.749 C 822 NH1 ARG 349 −5.488 −1.884 15.897 N 823 NH2 ARG 349 −7.355 −1.502 14.731 N1+ 824 C ARG 349 −3.189 0.016 9.838 C 825 O ARG 349 −2.579 0.975 10.31 O 826 H ARG 349 −6.142 −0.531 11.064 H 827 HA ARG 349 −3.993 −0.354 11.739 H 828 HB2 ARG 349 −3.339 −2.74 9.987 H 829 HB3 ARG 349 −2.352 −2.065 11.265 H 830 HG2 ARG 349 −4.953 −3.585 11.579 H 831 HG3 ARG 349 −3.329 −4.078 12.016 H 832 HD2 ARG 349 −4.047 −3.526 14.072 H 833 HD3 ARG 349 −3.401 −1.945 13.605 H 834 HE ARG 349 −6.169 −2.21 12.831 H 835 HH11 ARG 349 −5.973 −1.678 16.757 H 836 HH12 ARG 349 −4.493 −2.11 15.914 H 837 HH21 ARG 349 −7.819 −1.437 13.828 H 838 HH22 ARG 349 −7.895 −1.435 15.593 H 839 N VAL 350 −3.174 −0.269 8.537 N 840 CA VAL 350 −2.395 0.478 7.537 C 841 CB VAL 350 −2.142 −0.371 6.272 C 842 CG1 VAL 350 −3.373 −0.501 5.372 C 843 CG2 VAL 350 −0.988 0.16 5.414 C 844 C VAL 350 −3.044 1.827 7.221 C 845 O VAL 350 −4.263 1.99 7.304 O 846 H VAL 350 −3.791 −1.009 8.234 H 847 HA VAL 350 −1.421 0.68 7.984 H 848 HB VAL 350 −1.865 −1.375 6.597 H 849 HG11 VAL 350 −3.155 −1.196 4.562 H 850 HG12 VAL 350 −4.224 −0.85 5.951 H 851 HG13 VAL 350 −3.626 0.463 4.928 H 852 HG21 VAL 350 −0.762 −0.561 4.628 H 853 HG22 VAL 350 −1.261 1.101 4.938 H 854 HG23 VAL 350 −0.096 0.302 6.023 H 855 N GLN 351 −2.225 2.79 6.802 N 856 CA GLN 351 −2.669 4.092 6.319 C 857 CB GLN 351 −1.445 5.021 6.288 C 858 CG GLN 351 −1.74 6.437 5.768 C 859 CD GLN 351 −0.474 7.148 5.304 C 860 OE1 GLN 351 0.639 6.77 5.641 O 861 NE2 GLN 351 −0.584 8.111 4.42 N 862 C GLN 351 −3.329 3.967 4.932 C 863 O GLN 351 −2.636 3.847 3.919 O 864 H GLN 351 −1.24 2.583 6.742 H 865 HA GLN 351 −3.394 4.505 7.022 H 866 HB2 GLN 351 −1.022 5.098 7.291 H 867 HB3 GLN 351 −0.694 4.564 5.646 H 868 HG2 GLN 351 −2.415 6.389 4.917 H 869 HG3 GLN 351 −2.213 7.024 6.554 H 870 HE21 GLN 351 −1.491 8.368 4.041 H 871 HE22 GLN 351 0.267 8.567 4.101 H 872 N SER 352 −4.651 4.16 4.899 N 873 CA SER 352 −5.449 4.493 3.706 C 874 CB SER 352 −6.635 3.526 3.548 C 875 OG SER 352 −7.593 3.559 4.593 O 876 C SER 352 −5.84 5.986 3.644 C 877 O SER 352 −6.578 6.418 2.761 O 878 H SER 352 −5.144 4.145 5.788 H 879 HA SER 352 −4.818 4.33 2.833 H 880 HB2 SER 352 −7.15 3.761 2.618 H 881 HB3 SER 352 −6.244 2.51 3.471 H 882 HG SER 352 −7.151 3.6 5.467 H 883 N LYS 353 −5.284 6.8 4.552 N 884 CA LYS 353 −5.328 8.274 4.58 C 885 CB LYS 353 −5.023 8.719 6.023 C 886 CG LYS 353 −5.325 10.196 6.303 C 887 CD LYS 353 −5.05 10.529 7.774 C 888 CE LYS 353 −5.543 11.943 8.093 C 889 NZ LYS 353 −5.4 12.238 9.536 N1+ 890 C LYS 353 −4.343 8.874 3.56 C 891 O LYS 353 −3.263 8.31 3.385 O 892 H LYS 353 −4.689 6.352 5.229 H 893 HA LYS 353 −6.335 8.599 4.32 H 894 HB2 LYS 353 −5.631 8.118 6.704 H 895 HB3 LYS 353 −3.973 8.525 6.243 H 896 HG2 LYS 353 −4.702 10.828 5.671 H 897 HG3 LYS 353 −6.376 10.388 6.09 H 898 HD2 LYS 353 −5.576 9.815 8.41 H 899 HD3 LYS 353 −3.978 10.456 7.971 H 900 HE2 LYS 353 −4.967 12.663 7.504 H 901 HE3 LYS 353 −6.596 12.018 7.808 H 902 HZ1 LYS 353 −4.422 12.208 9.81 H 903 HZ2 LYS 353 −5.776 13.148 9.767 H 904 HZ3 LYS 353 −5.909 11.559 10.093 H 905 N ILE 354 −4.669 10.012 2.933 N 906 CA ILE 354 −3.851 10.612 1.854 C 907 CB ILE 354 −4.49 11.857 1.198 C 908 CG2 ILE 354 −5.875 11.535 0.615 C 909 CG1 ILE 354 −4.473 13.148 2.056 C 910 CD1 ILE 354 −5.382 13.198 3.288 C 911 C ILE 354 −2.397 10.916 2.255 C 912 O ILE 354 −2.127 11.249 3.414 O 913 H ILE 354 −5.559 10.441 3.152 H 914 HA ILE 354 −3.8 9.866 1.068 H 915 HB ILE 354 −3.864 12.081 0.333 H 916 HG12 ILE 354 −3.452 13.353 2.377 H 917 HG13 ILE 354 −4.754 13.977 1.414 H 918 HG21 ILE 354 −6.223 12.374 0.012 H 919 HG22 ILE 354 −5.808 10.66 −0.032 H 920 HG23 ILE 354 −6.598 11.338 1.403 H 921 HD11 ILE 354 −6.424 13.09 2.992 H 922 HD12 ILE 354 −5.109 12.417 3.994 H 923 HD13 ILE 354 −5.263 14.164 3.78 H 924 N GLY 355 −1.48 10.871 1.283 N 925 CA GLY 355 −0.044 11.093 1.498 C 926 C GLY 355 0.813 11.379 0.253 C 927 O GLY 355 1.931 11.867 0.409 O 928 H GLY 355 −1.791 10.604 0.35 H 929 HA2 GLY 355 0.087 11.931 2.183 H 930 HA3 GLY 355 0.367 10.205 1.976 H 931 N SER 356 0.326 11.166 −0.98 N 932 CA SER 356 1.095 11.523 −2.191 C 933 CB SER 356 0.55 10.785 −3.423 C 934 OG SER 356 1.168 11.154 −4.653 O 935 C SER 356 1.148 13.048 −2.386 C 936 O SER 356 0.121 13.734 −2.408 O 937 H SER 356 −0.62 10.805 −1.092 H 938 HA SER 356 2.117 11.173 −2.047 H 939 HB2 SER 356 0.676 9.712 −3.272 H 940 HB3 SER 356 −0.518 10.991 −3.505 H 941 HG SER 356 2.148 11.161 −4.615 H 942 N LEU 357 2.368 13.58 −2.513 N 943 CA LEU 357 2.681 15.005 −2.714 C 944 CB LEU 357 3.865 15.363 −1.797 C 945 CG LEU 357 3.456 15.585 −0.332 C 946 CD1 LEU 357 4.702 15.569 0.547 C 947 CD2 LEU 357 2.774 16.947 −0.165 C 948 C LEU 357 3.014 15.36 −4.172 C 949 O LEU 357 3.038 16.535 −4.536 O 950 H LEU 357 3.151 12.946 −2.445 H 951 HA LEU 357 1.82 15.614 −2.443 H 952 HB2 LEU 357 4.607 14.564 −1.857 H 953 HB3 LEU 357 4.344 16.274 −2.16 H 954 HG LEU 357 2.783 14.794 −0.003 H 955 HD11 LEU 357 5.219 14.615 0.439 H 956 HD12 LEU 357 5.368 16.373 0.245 H 957 HD13 LEU 357 4.425 15.689 1.591 H 958 HD21 LEU 357 1.859 16.989 −0.752 H 959 HD22 LEU 357 2.514 17.102 0.88 H 960 HD23 LEU 357 3.442 17.748 −0.482 H 961 N ASP 358 3.303 14.349 −4.986 N 962 CA ASP 358 3.683 14.447 −6.389 C 963 CB ASP 358 4.644 13.293 −6.744 C 964 CG ASP 358 4.224 11.87 −6.317 C 965 OD1 ASP 358 3.854 11.658 −5.128 O 966 OD2 ASP 358 4.43 10.953 −7.145 O1− 967 C ASP 358 2.457 14.546 −7.315 C 968 O ASP 358 1.51 13.764 −7.211 O 969 H ASP 358 3.25 13.404 −4.62 H 970 HA ASP 358 4.251 15.369 −6.515 H 971 HB2 ASP 358 4.817 13.312 −7.821 H 972 HB3 ASP 358 5.601 13.503 −6.265 H 973 N ASN 359 2.466 15.535 −8.218 N 974 CA ASN 359 1.357 15.825 −9.133 C 975 CB ASN 359 1.304 17.339 −9.42 C 976 CG ASN 359 0.404 18.088 −8.452 C 977 OD1 ASN 359 −0.771 18.311 −8.703 O 978 ND2 ASN 359 0.892 18.555 −7.329 N 979 C ASN 359 1.435 14.961 −10.41 C 980 O ASN 359 2.465 14.966 −11.099 O 981 H ASN 359 3.292 16.11 −8.293 H 982 HA ASN 359 0.422 15.573 −8.636 H 983 HB2 ASN 359 2.302 17.775 −9.41 H 984 HB3 ASN 359 0.884 17.495 −10.411 H 985 HD21 ASN 359 1.865 18.431 −7.069 H 986 HD22 ASN 359 0.262 19.03 −6.706 H 987 N ILE 360 0.343 14.252 −10.723 N 988 CA ILE 360 0.171 13.307 −11.839 C 989 CB ILE 360 −0.11 11.882 −11.291 C 990 CG2 ILE 360 −0.243 10.847 −12.425 C 991 CG1 ILE 360 0.928 11.383 −10.254 C 992 CD1 ILE 360 2.384 11.316 −10.734 C 993 C ILE 360 −0.973 13.781 −12.753 C 994 O ILE 360 −2.08 14.055 −12.285 O 995 H ILE 360 −0.431 14.298 −10.065 H 996 HA ILE 360 1.083 13.273 −12.43 H 997 HB ILE 360 −1.072 11.913 −10.775 H 998 HG12 ILE 360 0.893 12.028 −9.376 H 999 HG13 ILE 360 0.634 10.388 −9.918 H 1000 HG21 ILE 360 0.665 10.816 −13.025 H 1001 HG22 ILE 360 −0.432 9.858 −12.006 H 1002 HG23 ILE 360 −1.084 11.099 −13.072 H 1003 HD11 ILE 360 3.01 10.968 −9.911 H 1004 HD12 ILE 360 2.482 10.626 −11.572 H 1005 HD13 ILE 360 2.732 12.303 −11.029 H 1006 N THR 361 −0.732 13.813 −14.067 N 1007 CA THR 361 −1.736 14.205 −15.07 C 1008 CB THR 361 −1.123 15.099 −16.159 C 1009 CG2 THR 361 −0.147 14.413 −17.113 C 1010 OG1 THR 361 −2.132 15.696 −16.94 O 1011 C THR 361 −2.475 12.989 −15.638 C 1012 O THR 361 −1.898 11.909 −15.774 O 1013 H THR 361 0.136 13.412 −14.402 H 1014 HA THR 361 −2.48 14.819 −14.566 H 1015 HB THR 361 −0.584 15.905 −15.665 H 1016 HG21 THR 361 0.175 15.122 −17.876 H 1017 HG22 THR 361 0.73 14.082 −16.561 H 1018 HG23 THR 361 −0.621 13.557 −17.591 H 1019 HG1 THR 361 −2.288 16.567 −16.525 H 1020 N HID 362 −3.756 13.163 −15.971 N 1021 CA HID 362 −4.655 12.082 −16.386 C 1022 CB HID 362 −6.101 12.601 −16.468 C 1023 CG HID 362 −7.149 11.514 −16.434 C 1024 ND1 HID 362 −7.928 11.192 −15.352 N 1025 CE1 HID 362 −8.703 10.149 −15.679 C 1026 NE2 HID 362 −8.512 9.811 −16.968 N 1027 CD2 HID 362 −7.516 10.672 −17.448 C 1028 C HID 362 −4.239 11.448 −17.722 C 1029 O HID 362 −4.035 12.155 −18.72 O 1030 H HID 362 −4.148 14.086 −15.826 H 1031 HA HID 362 −4.615 11.317 −15.613 H 1032 HB2 HID 362 −6.285 13.264 −15.626 H 1033 HB3 HID 362 −6.225 13.179 −17.383 H 1034 HD1 HID 362 −7.953 11.711 −14.475 H 1035 HD2 HID 362 −7.102 10.674 −18.444 H 1036 HE1 HID 362 −9.412 9.672 −15.012 H 1037 N VAL 363 −4.258 10.114 −17.788 N 1038 CA VAL 363 −4.047 9.333 −19.019 C 1039 CB VAL 363 −2.59 8.821 −19.102 C 1040 CG1 VAL 363 −2.325 8.173 −20.469 C 1041 CG2 VAL 363 −1.535 9.924 −18.926 C 1042 C VAL 363 −5.063 8.172 −19.047 C 1043 O VAL 363 −4.937 7.244 −18.247 O 1044 H VAL 363 −4.416 9.604 −16.913 H 1045 HA VAL 363 −4.2 9.973 −19.884 H 1046 HB VAL 363 −2.423 8.087 −18.315 H 1047 HG11 VAL 363 −1.31 7.773 −20.496 H 1048 HG12 VAL 363 −3.016 7.351 −20.645 H 1049 HG13 VAL 363 −2.439 8.911 −21.264 H 1050 HG21 VAL 363 −0.539 9.503 −19.053 H 1051 HG22 VAL 363 −1.695 10.716 −19.655 H 1052 HG23 VAL 363 −1.595 10.341 −17.92 H 1053 N PRO 364 −6.102 8.176 −19.914 N 1054 CD PRO 364 −6.471 9.247 −20.831 C 1055 CG PRO 364 −7.988 9.152 −20.982 C 1056 CB PRO 364 −8.234 7.651 −20.878 C 1057 CA PRO 364 −7.198 7.19 −19.842 C 1058 C PRO 364 −6.75 5.738 −20.083 C 1059 O PRO 364 −7.006 4.848 −19.265 O 1060 HA PRO 364 −7.651 7.252 −18.853 H 1061 HB2 PRO 364 −8.033 7.201 −21.851 H 1062 HB3 PRO 364 −9.253 7.429 −20.559 H 1063 HG2 PRO 364 −8.326 9.556 −21.936 H 1064 HG3 PRO 364 −8.48 9.66 −20.151 H 1065 HD2 PRO 364 −5.99 9.082 −21.797 H 1066 HD3 PRO 364 −6.207 10.231 −20.446 H 1067 N GLY 365 −5.962 5.529 −21.142 N 1068 CA GLY 365 −5.241 4.284 −21.426 C 1069 C GLY 365 −3.864 4.235 −20.752 C 1070 O GLY 365 −2.876 3.901 −21.409 O 1071 H GLY 365 −5.792 6.314 −21.755 H 1072 HA2 GLY 365 −5.827 3.434 −21.076 H 1073 HA3 GLY 365 −5.115 4.18 −22.501 H 1074 N GLY 366 −3.774 4.761 −19.526 N 1075 CA GLY 366 −2.551 4.949 −18.742 C 1076 C GLY 366 −2.68 4.471 −17.291 C 1077 O GLY 366 −3.709 3.918 −16.893 O 1078 H GLY 366 −4.63 5.077 −19.091 H 1079 HA2 GLY 366 −1.719 4.421 −19.208 H 1080 HA3 GLY 366 −2.291 6.002 −18.724 H 1081 N GLY 367 −1.573 4.554 −16.559 N 1082 CA GLY 367 −1.345 3.827 −15.313 C 1083 C GLY 367 −2.028 4.421 −14.081 C 1084 O GLY 367 −2.552 5.533 −14.094 O 1085 H GLY 367 −0.793 5.059 −16.962 H 1086 HA2 GLY 367 −1.706 2.807 −15.437 H 1087 HA3 GLY 367 −0.273 3.777 −15.117 H 1088 N ASN 368 −1.948 3.675 −12.983 N 1089 CA ASN 368 −2.581 4.026 −11.715 C 1090 CB ASN 368 −2.83 2.71 −10.954 C 1091 CG ASN 368 −3.89 1.826 −11.598 C 1092 OD1 ASN 368 −4.537 2.16 −12.581 O 1093 ND2 ASN 368 −4.129 0.654 −11.066 N 1094 C ASN 368 −1.748 5.055 −10.915 C 1095 O ASN 368 −0.525 4.909 −10.81 O 1096 H ASN 368 −1.518 2.76 −13.065 H 1097 HA ASN 368 −3.546 4.487 −11.932 H 1098 HB2 ASN 368 −1.901 2.145 −10.882 H 1099 HB3 ASN 368 −3.154 2.935 −9.944 H 1100 HD21 ASN 368 −3.56 0.253 −10.33 H 1101 HD22 ASN 368 −4.885 0.124 −11.48 H 1102 N LYS 369 −2.401 6.049 −10.292 N 1103 CA LYS 369 −1.797 7.102 −9.44 C 1104 CB LYS 369 −2.864 8.168 −9.11 C 1105 CG LYS 369 −4.001 7.735 −8.148 C 1106 CD LYS 369 −3.729 7.865 −6.637 C 1107 CE LYS 369 −3.436 9.312 −6.232 C 1108 NZ LYS 369 −2.751 9.419 −4.929 N1+ 1109 C LYS 369 −1.119 6.538 −8.183 C 1110 O LYS 369 −1.519 5.475 −7.707 O 1111 H LYS 369 −3.408 6.088 −10.427 H 1112 HA LYS 369 −1.017 7.588 −10.027 H 1113 HB2 LYS 369 −2.365 9.056 −8.729 H 1114 HB3 LYS 369 −3.328 8.466 −10.05 H 1115 HG2 LYS 369 −4.882 8.334 −8.379 H 1116 HG3 LYS 369 −4.259 6.697 −8.348 H 1117 HD2 LYS 369 −4.607 7.521 −6.088 H 1118 HD3 LYS 369 −2.897 7.226 −6.362 H 1119 HE2 LYS 369 −2.799 9.769 −6.989 H 1120 HE3 LYS 369 −4.373 9.873 −6.206 H 1121 HZ LYS 369 −2.542 10.391 −4.717 H 1122 HZ2 LYS 369 −3.344 9.102 −4.164 H 1123 HZ3 LYS 369 −1.889 8.882 −4.921 H 1124 N LYS 370 −0.136 7.236 −7.594 N 1125 CA LYS 370 0.67 6.677 −6.488 C 1126 CB LYS 370 1.914 7.544 −6.193 C 1127 CG LYS 370 2.78 6.887 −5.1 C 1128 CD LYS 370 4.099 7.614 −4.813 C 1129 CE LYS 370 4.794 7 −3.584 C 1130 NZ LYS 370 5.236 5.6 −3.818 N1+ 1131 C LYS 370 −0.169 6.426 −5.228 C 1132 O LYS 370 −0.9 7.306 −4.767 O 1133 H LYS 370 0.049 8.184 −7.904 H 1134 HA LYS 370 1.032 5.704 −6.827 H 1135 HB2 LYS 370 2.505 7.648 −7.104 H 1136 HB3 LYS 370 1.604 8.536 −5.86 H 1137 HG2 LYS 370 2.214 6.868 −4.17 H 1138 HG3 LYS 370 2.999 5.86 −5.396 H 1139 HD2 LYS 370 4.754 7.562 −5.685 H 1140 HD3 LYS 370 3.889 8.665 −4.605 H 1141 HE2 LYS 370 5.663 7.618 −3.339 H 1142 HE3 LYS 370 4.102 7.038 −2.736 H 1143 HZ1 LYS 370 5.673 5.196 −2.99 H 1144 HZ2 LYS 370 4.462 4.98 −4.047 H 1145 HZ3 LYS 370 5.92 5.574 −4.571 H 1146 N ILE 371 −0.009 5.232 −4.649 N 1147 CA ILE 371 −0.668 4.808 −3.405 C 1148 CB ILE 371 −0.386 3.313 −3.105 C 1149 CG2 ILE 371 −1.069 2.871 −1.796 C 1150 CG1 ILE 371 −0.84 2.413 −4.282 C 1151 CD1 ILE 371 −0.501 0.926 −4.115 C 1152 C ILE 371 −0.244 5.722 −2.244 C 1153 O ILE 371 0.936 6.025 −2.058 O 1154 H ILE 371 0.615 4.577 −5.091 H 1155 HA ILE 371 −1.744 4.92 −3.545 H 1156 HB ILE 371 0.689 3.192 −2.978 H 1157 HG12 ILE 371 −1.917 2.515 −4.424 H 1158 HG13 ILE 371 −0.349 2.742 −5.197 H 1159 HG21 ILE 371 −0.812 1.838 −1.562 H 1160 HG22 ILE 371 −0.718 3.465 −0.954 H 1161 HG23 ILE 371 −2.153 2.957 −1.886 H 1162 HD11 ILE 371 0.55 0.815 −3.859 H 1163 HD12 ILE 371 −1.114 0.475 −3.336 H 1164 HD13 ILE 371 −0.691 0.403 −5.053 H 1165 N GLU 372 −1.229 6.158 −1.458 N 1166 CA GLU 372 −1.084 7.207 −0.439 C 1167 CB GLU 372 −2.468 7.488 0.17 C 1168 CG GLU 372 −3.568 7.904 −0.826 C 1169 CD GLU 372 −3.292 9.211 −1.583 C 1170 OE1 GLU 372 −4.048 9.506 −2.539 O 1171 OE2 GLU 372 −2.324 9.933 −1.254 O1− 1172 C GLU 372 −0.118 6.846 0.701 C 1173 O GLU 372 0.622 7.706 1.177 O 1174 H GLU 372 −2.168 5.871 −1.688 H 1175 HA GLU 372 −0.705 8.116 −0.907 H 1176 HB2 GLU 372 −2.81 6.589 0.685 H 1177 HB3 GLU 372 −2.352 8.265 0.919 H 1178 HG2 GLU 372 −3.72 7.097 −1.546 H 1179 HG3 GLU 372 −4.499 8.017 −0.268 H 1180 N THR 373 −0.103 5.569 1.094 N 1181 CA THR 373 0.553 5.012 2.286 C 1182 CB THR 373 0.449 3.477 2.216 C 1183 CG2 THR 373 0.91 2.759 3.483 C 1184 OG1 THR 373 −0.889 3.093 1.969 O 1185 C THR 373 2.024 5.425 2.421 C 1186 O THR 373 2.856 5.061 1.59 O 1187 H THR 373 −0.708 4.933 0.597 H 1188 HA THR 373 0.012 5.353 3.167 H 1189 HB THR 373 1.055 3.123 1.381 H 1190 HG21 THR 373 0.352 3.114 4.345 H 1191 HG22 THR 373 0.746 1.689 3.366 H 1192 HG23 THR 373 1.975 2.929 3.64 H 1193 HG1 THR 373 −1.437 3.366 2.731 H 1194 N HIE 374 2.371 6.118 3.511 N 1195 CA HIE 374 3.731 6.573 3.845 C 1196 CB HIE 374 3.852 8.084 3.572 C 1197 CG HIE 374 3.066 8.981 4.497 C 1198 ND1 HIE 374 1.949 9.734 4.132 N 1199 CE1 HIE 374 1.625 10.465 5.209 C 1200 NE2 HIE 374 2.473 10.21 6.219 N 1201 CD2 HIE 374 3.391 9.279 5.788 C 1202 C HIE 374 4.205 6.174 5.257 C 1203 O HIE 374 5.304 6.564 5.662 O 1204 H HIE 374 1.627 6.449 4.119 H 1205 HA HIE 374 4.433 6.08 3.172 H 1206 HB2 HIE 374 4.902 8.37 3.637 H 1207 HB3 HIE 374 3.529 8.283 2.55 H 1208 HD2 HIE 374 4.239 8.894 6.337 H 1209 HE2 HIE 374 2.458 10.661 7.123 H 1210 HE1 HIE 374 0.803 11.167 5.252 H 1211 N LYS 375 3.428 5.35 5.978 N 1212 CA LYS 375 3.809 4.689 7.241 C 1213 CB LYS 375 2.845 5.081 8.379 C 1214 CG LYS 375 2.848 6.582 8.714 C 1215 CD LYS 375 2.415 6.805 10.173 C 1216 CE LYS 375 2.116 8.282 10.429 C 1217 NZ LYS 375 2.008 8.598 11.874 N1+ 1218 C LYS 375 3.91 3.16 7.117 C 1219 O LYS 375 3.457 2.563 6.138 O 1220 H LYS 375 2.515 5.137 5.601 H 1221 HA LYS 375 4.808 5.027 7.515 H 1222 HB2 LYS 375 1.828 4.777 8.121 H 1223 HB3 LYS 375 3.135 4.532 9.276 H 1224 HG2 LYS 375 3.852 6.99 8.585 H 1225 HG3 LYS 375 2.168 7.1 8.036 H 1226 HD2 LYS 375 1.519 6.22 10.384 H 1227 HD3 LYS 375 3.218 6.474 10.833 H 1228 HE2 LYS 375 2.915 8.89 9.994 H 1229 HE3 LYS 375 1.183 8.541 9.922 H 1230 HZ1 LYS 375 1.29 8.048 12.343 H 1231 HZ2 LYS 375 2.908 8.477 12.335 H 1232 HZ3 LYS 375 1.803 9.591 11.966 H 1233 N LEU 376 4.489 2.536 8.144 N 1234 CA LEU 376 4.687 1.087 8.281 C 1235 CB LEU 376 6.082 0.846 8.892 C 1236 CG LEU 376 7.246 1.519 8.133 C 1237 CD1 LEU 376 8.548 1.284 8.892 C 1238 CD2 LEU 376 7.379 0.987 6.705 C 1239 C LEU 376 3.577 0.437 9.133 C 1240 O LEU 376 3.058 1.078 10.047 O 1241 H LEU 376 4.852 3.123 8.887 H 1242 HA LEU 376 4.657 0.626 7.293 H 1243 HB2 LEU 376 6.076 1.227 9.915 H 1244 HB3 LEU 376 6.263 −0.229 8.937 H 1245 HG LEU 376 7.089 2.596 8.084 H 1246 HD11 LEU 376 8.496 1.771 9.866 H 1247 HD12 LEU 376 8.711 0.222 9.032 H 1248 HD13 LEU 376 9.383 1.713 8.338 H 1249 HD21 LEU 376 7.445 −0.099 6.706 H 1250 HD22 LEU 376 6.523 1.3 6.109 H 1251 HD23 LEU 376 8.277 1.403 6.248 H 1252 N THR 377 3.229 −0.833 8.87 N 1253 CA THR 377 2.053 −1.515 9.479 C 1254 CB THR 377 0.91 −1.594 8.449 C 1255 CG2 THR 377 1.154 −2.619 7.341 C 1256 OG1 THR 377 −0.31 −1.923 9.066 O 1257 C THR 377 2.318 −2.896 10.115 C 1258 O THR 377 1.376 −3.562 10.552 O 1259 H THR 377 3.681 −1.281 8.085 H 1260 HA THR 377 1.683 −0.894 10.296 H 1261 HB THR 377 0.797 −0.612 7.99 H 1262 HG21 THR 377 1.137 −3.633 7.743 H 1263 HG22 THR 377 0.372 −2.529 6.587 H 1264 HG23 THR 377 2.119 −2.437 6.871 H 1265 HG1 THR 377 −0.127 −2.677 9.648 H 1266 N PHE 378 3.572 −3.356 10.153 N 1267 CA PHE 378 3.952 −4.685 10.657 C 1268 CB PHE 378 4.648 −5.479 9.541 C 1269 CG PHE 378 3.813 −5.665 8.288 C 1270 CD1 PHE 378 2.693 −6.517 8.313 C 1271 CE1 PHE 378 1.923 −6.708 7.153 C 1272 CZ PHE 378 2.273 −6.054 5.96 C 1273 CE2 PHE 378 3.389 −5.199 5.932 C 1274 CD2 PHE 378 4.153 −4.997 7.095 C 1275 C PHE 378 4.852 −4.58 11.894 C 1276 O PHE 378 5.662 −3.654 11.983 O 1277 H PHE 378 4.315 −2.755 9.833 H 1278 HA PHE 378 3.059 −5.238 10.947 H 1279 HB2 PHE 378 5.58 −4.976 9.28 H 1280 HB3 PHE 378 4.909 −6.467 9.924 H 1281 HD1 PHE 378 2.42 −7.028 9.225 H 1282 HD2 PHE 378 5.021 −4.353 7.063 H 1283 HE1 PHE 378 1.059 −7.359 7.184 H 1284 HE2 PHE 378 3.67 −4.715 5.009 H 1285 HZ PHE 378 1.683 −6.212 5.067 H 1286 N ARG 379 4.757 −5.538 12.826 N 1287 CA ARG 379 5.738 −5.681 13.92 C 1288 CB ARG 379 5.147 −6.446 15.123 C 1289 CG ARG 379 5.333 −7.972 15.132 C 1290 CD ARG 379 4.674 −8.726 13.972 C 1291 NE ARG 379 4.809 −10.17 14.201 N 1292 CZ ARG 379 3.997 −11.122 13.79 C 1293 NH1 ARG 379 3.16 −10.997 12.8 N 1294 NH2 ARG 379 4.005 −12.259 14.409 N1+ 1295 C ARG 379 7.065 −6.248 13.403 C 1296 O ARG 379 7.099 −6.892 12.357 O 1297 H ARG 379 4.046 −6.244 12.717 H 1298 HA ARG 379 5.968 −4.677 14.283 H 1299 HB2 ARG 379 5.635 −6.067 16.023 H 1300 HB3 ARG 379 4.085 −6.213 15.22 H 1301 HG2 ARG 379 6.398 −8.205 15.144 H 1302 HG3 ARG 379 4.912 −8.346 16.065 H 1303 HD2 ARG 379 3.617 −8.456 13.932 H 1304 HD3 ARG 379 5.153 −8.456 13.03 H 1305 HE ARG 379 5.536 −10.466 14.839 H 1306 HH11 ARG 379 2.607 −11.804 12.542 H 1307 HH12 ARG 379 3.237 −10.214 12.165 H 1308 HH21 ARG 379 3.368 −12.977 14.092 H 1309 HH22 ARG 379 4.409 −12.292 15.336 H 1310 N GLU 380 8.158 −6.012 14.117 N 1311 CA GLU 380 9.531 −6.247 13.64 C 1312 CB GLU 380 10.473 −5.223 14.308 C 1313 CG GLU 380 10.918 −5.532 15.752 C 1314 CD GLU 380 9.776 −5.749 16.759 C 1315 OE1 GLU 380 8.713 −5.095 16.645 O 1316 OE2 GLU 380 9.949 −6.589 17.669 O1− 1317 C GLU 380 10.05 −7.688 13.804 C 1318 O GLU 380 11.068 −8.049 13.211 O 1319 H GLU 380 8.058 −5.508 14.999 H 1320 HA GLU 380 9.553 −6.041 12.569 H 1321 HB2 GLU 380 11.376 −5.156 13.698 H 1322 HB3 GLU 380 10.002 −4.239 14.283 H 1323 HG2 GLU 380 11.554 −6.42 15.734 H 1324 HG3 GLU 380 11.536 −4.703 16.101 H 1325 N ASN 381 9.378 −8.507 14.619 N 1326 CA ASN 381 9.888 −9.793 15.1 C 1327 CB ASN 381 10.277 −9.59 16.576 C 1328 CG ASN 381 10.946 −10.77 17.253 C 1329 OD1 ASN 381 11.18 −11.824 16.676 O 1330 ND2 ASN 381 11.308 −10.609 18.503 N 1331 C ASN 381 8.886 −10.941 14.87 C 1332 C ASN 381 7.697 −10.829 15.186 O 1333 H ASN 381 8.556 −8.138 15.073 H 1334 HA ASN 381 10.798 −10.044 14.553 H 1335 HB2 ASN 381 10.977 −8.756 16.634 H 1336 HB3 ASN 381 9.395 −9.315 17.151 H 1337 HD21 ASN 381 11.177 −9.696 18.934 H 1338 HD22 ASN 381 11.842 −11.336 18.94 H 1339 N ALA 382 9.396 −12.078 14.381 N 1340 CA ALA 382 8.625 −13.265 14.012 C 1341 CB ALA 382 9.612 −14.306 13.473 C 1342 C ALA 382 7.778 −13.868 15.151 C 1343 O ALA 382 6.683 −14.383 14.897 O 1344 H ALA 382 10.388 −12.099 14.187 H 1345 HA ALA 382 7.945 −12.991 13.209 H 1346 HB1 ALA 382 10.141 −13.902 12.61 H 1347 HB2 ALA 382 10.336 −14.576 14.244 H 1348 HB3 ALA 382 9.069 −15.202 13.169 H 1349 N LYS 383 8.247 −13.783 16.404 N 1350 CA LYS 383 7.517 −14.292 17.581 C 1351 CB LYS 383 8.48 −15.014 18.536 C 1352 CG LYS 383 9.477 −14.065 19.214 C 1353 CD LYS 383 10.385 −14.833 20.177 C 1354 CE LYS 383 11.402 −13.86 20.769 C 1355 NZ LYS 383 12.327 −14.546 21.691 N1+ 1356 C LYS 383 6.653 −13.25 18.302 C 1357 O LYS 383 5.815 −13.633 19.116 O 1358 H LYS 383 9.171 −13.385 16.536 H 1359 HA LYS 383 6.81 −15.048 17.242 H 1360 HB2 LYS 383 7.895 −15.52 19.306 H 1361 HB3 LYS 383 9.029 −15.776 17.979 H 1362 HG2 LYS 383 10.089 −13.58 18.453 H 1363 HG3 LYS 383 8.939 −13.299 19.774 H 1364 HD2 LYS 383 9.782 −15.271 20.974 H 1365 HD3 LYS 383 10.904 −15.629 19.64 H 1366 HE2 LYS 383 11.971 −13.406 19.952 H 1367 HE3 LYS 383 10.864 −13.072 21.304 H 1368 HZ1 LYS 383 11.819 −15.028 22.429 H 1369 HZ2 LYS 383 12.919 −15.203 21.189 H 1370 HZ3 LYS 383 12.967 −13.878 22.111 H 1371 N ALA 384 6.825 −11.959 18.006 N 1372 CA ALA 384 6.026 −10.883 18.599 C 1373 CB ALA 384 6.716 −9.542 18.323 C 1374 C ALA 384 4.566 −10.924 18.104 C 1375 O ALA 384 4.267 −11.56 17.087 O 1376 H ALA 384 7.463 −11.716 17.263 H 1377 HA ALA 384 6.004 −11.023 19.682 H 1378 HB1 ALA 384 7.689 −9.521 18.815 H 1379 HB2 ALA 384 6.847 −9.396 17.253 H 1380 HB3 ALA 384 6.114 −8.722 18.717 H 1381 N LYS 385 3.65 −10.274 18.833 N 1382 CA LYS 385 2.2 −10.289 18.553 C 1383 CB LYS 385 1.422 −10.295 19.885 C 1384 CG LYS 385 1.658 −11.611 20.647 C 1385 CD LYS 385 0.686 −11.789 21.82 C 1386 CE LYS 385 1.005 −13.101 22.549 C 1387 NZ LYS 385 −0.032 −13.429 23.551 N1+ 1388 C LYS 385 1.777 −9.151 17.612 C 1389 O LYS 385 2.583 −8.298 17.249 O 1390 H LYS 385 3.981 −9.678 19.586 H 1391 HA LYS 385 1.953 −11.207 18.02 H 1392 HB2 LYS 385 1.732 −9.449 20.502 H 1393 HB3 LYS 385 0.356 −10.194 19.683 H 1394 HG2 LYS 385 1.523 −12.448 19.96 H 1395 HG3 LYS 385 2.682 −11.631 21.024 H 1396 HD2 LYS 385 0.786 −10.95 22.513 H 1397 HD3 LYS 385 −0.336 −11.818 21.437 H 1398 HE2 LYS 385 1.069 −13.909 21.814 H 1399 HE3 LYS 385 1.979 −13.003 23.037 H 1400 HZ1 LYS 385 0.169 −14.3 24.032 H 1401 HZ2 LYS 385 −0.113 −12.692 24.251 H 1402 HZ3 LYS 385 −0.935 −13.568 23.102 H 1403 N THR 386 0.517 −9.161 17.17 N 1404 CA THR 386 −0.09 −8.058 16.412 C 1405 CB THR 386 0.078 −8.223 14.888 C 1406 CG2 THR 386 −0.675 −9.412 14.301 C 1407 OG1 THR 386 −0.344 −7.064 14.186 O 1408 C THR 386 −1.552 −7.835 16.787 C 1409 O THR 386 −2.302 −8.766 17.082 O 1410 H THR 386 −0.099 −9.909 17.474 H 1411 HA THR 386 0.445 −7.151 16.692 H 1412 HB THR 386 1.14 −8.363 14.683 H 1413 HG21 THR 386 −1.752 −9.287 14.423 H 1414 HG22 THR 386 −0.443 −9.497 13.239 H 1415 HG23 THR 386 −0.359 −10.323 14.806 H 1416 HG1 THR 386 −1.314 −7.006 14.253 H 1417 N ASP 387 −1.941 −6.568 16.724 N 1418 CA ASP 387 −3.299 −6.053 16.79 C 1419 CB ASP 387 −3.238 −4.574 17.233 C 1420 CG ASP 387 −2.35 −3.649 16.372 C 1421 OD1 ASP 387 −1.201 −4.019 16.023 O 1422 OD2 ASP 387 −2.791 −2.516 16.069 O1− 1423 C ASP 387 −4.053 −6.248 15.459 C 1424 O ASP 387 −3.456 −6.206 14.375 O 1425 H ASP 387 −1.25 −5.869 16.471 H 1426 HA ASP 387 −3.833 −6.606 17.56 H 1427 HB2 ASP 387 −4.255 −4.181 17.252 H 1428 HB3 ASP 387 −2.861 −4.541 18.257 H 1429 N HIE 388 −5.372 −6.462 15.536 N 1430 CA HIE 388 −6.281 −6.542 14.383 C 1431 CB HIE 388 −6.257 −7.967 13.771 C 1432 CG HIE 388 −7.094 −9.041 14.435 C 1433 ND1 HIE 388 −6.62 −10.102 15.22 N 1434 CE1 HIE 388 −7.692 −10.868 15.489 C 1435 NE2 HIE 388 −8.792 −10.359 14.913 N 1436 CD2 HIE 388 −8.43 −9.225 14.227 C 1437 C HIE 388 −7.704 −6.043 14.714 C 1438 O HIE 388 −8.036 −5.796 15.874 O 1439 H HIE 388 −5.795 −6.52 16.46 H 1440 HA HIE 388 −5.898 −5.861 13.622 H 1441 HB2 HIE 388 −6.605 −7.891 12.741 H 1442 HB3 HIE 388 −5.225 −8.317 13.724 H 1443 HD2 HIE 388 −9.071 −8.617 13.606 H 1444 HE2 HIE 388 −9.73 −10.731 15.001 H 1445 HE1 HIE 388 −7.681 −11.76 16.103 H 1446 N GLY 389 −8.534 −5.88 13.678 N 1447 CA GLY 389 −9.949 −5.486 13.737 C 1448 C GLY 389 −10.872 −6.603 14.242 C 1449 O GLY 389 −10.628 −7.171 15.307 O 1450 H GLY 389 −8.146 −6.019 12.752 H 1451 HA2 GLY 389 −10.058 −4.624 14.392 H 1452 HA3 GLY 389 −10.271 −5.185 12.739 H 1453 N ALA 390 −11.933 −6.934 13.5 N 1454 CA ALA 390 −12.845 −8.038 13.829 C 1455 CB ALA 390 −14.197 −7.776 13.152 C 1456 C ALA 390 −12.289 −9.444 13.487 C 1457 O ALA 390 −11.409 −9.613 12.632 O 1458 H ALA 390 −12.141 −6.386 12.672 H 1459 HA ALA 390 −13.018 −8.019 14.907 H 1460 HB1 ALA 390 −14.083 −7.8 12.069 H 1461 HB2 ALA 390 −14.916 −8.54 13.45 H 1462 HB3 ALA 390 −14.576 −6.797 13.451 H 1463 N GLU 391 −12.83 −10.471 14.157 N 1464 CA GLU 391 −12.433 −11.891 14.063 C 1465 CB GLU 391 −11.893 −12.318 15.437 C 1466 CG GLU 391 −11.31 −13.733 15.478 C 1467 CD GLU 391 −9.913 −13.835 14.866 C 1468 OE1 GLU 391 −8.972 −14.255 15.579 O 1469 OE2 GLU 391 −9.749 −13.547 13.653 O1− 1470 C GLU 391 −13.535 −12.84 13.56 C 1471 O GLU 391 −14.497 −13.128 14.308 O 1472 OXT GLU 391 −13.391 −13.329 12.418 O1− 1473 H GLU 391 −13.501 −10.247 14.883 H 1474 HA GLU 391 −11.631 −11.983 13.337 H 1475 HB2 GLU 391 −11.124 −11.618 15.763 H 1476 HB3 GLU 391 −12.709 −12.27 16.157 H 1477 HG2 GLU 391 −11.272 −14.031 16.525 H 1478 HG3 GLU 391 −11.964 −14.429 14.955 H 1479 C8 MOL 392 −6.578 2.271 0.481 C 1480 C6 MOL 392 −5.466 1.518 0.847 C 1481 S MOL 392 −5.715 −0.133 1.394 S 1482 C3 MOL 392 −4.098 −0.81 1.481 C 1483 C2 MOL 392 −3.96 −2.171 1.736 C 1484 C1 MOL 392 −2.688 −2.745 1.888 C 1485 N1 MOL 392 −2.576 −4.13 2.254 N 1486 C12 MOL 392 −3.569 −5.055 1.658 C 1487 C13 MOL 392 −1.228 −4.747 2.289 C 1488 C MOL 392 −1.552 −1.945 1.713 C 1489 C5 MOL 392 −1.685 −0.587 1.43 C 1490 C4 MOL 392 −2.947 −0.018 1.323 C 1491 N MOL 392 −3.009 1.369 1.055 N 1492 C7 MOL 392 −4.186 2.093 0.757 C 1493 C11 MOL 392 −4.036 3.412 0.346 C 1494 C10 MOL 392 −5.15 4.157 −0.034 C 1495 C9 MOL 392 −6.425 3.587 0.017 C 1496 N2 MOL 392 −7.541 4.346 −0.45 N 1497 C15 MOL 392 −7.696 5.732 0.055 C 1498 C14 MOL 392 −8.867 3.704 −0.53 C 1499 H1 MOL 392 −7.532 1.832 0.559 H 1500 H3 MOL 392 −4.829 −2.76 1.839 H 1501 H7 MOL 392 −3.583 −4.88 0.59 H 1502 H8 MOL 392 −4.533 −4.861 2.106 H 1503 H9 MOL 392 −3.283 −6.077 1.87 H 1504 H10 MOL 392 −1.32 −5.764 2.647 H 1505 H11 MOL 392 −0.824 −4.728 1.284 H 1506 H12 MOL 392 −0.617 −4.181 2.978 H 1507 H2 MOL 392 −0.584 −2.357 1.781 H 1508 H4 MOL 392 −0.824 0.018 1.292 H 1509 H MOL 392 −2.124 1.911 1.149 H 1510 H6 MOL 392 −3.07 3.845 0.31 H 1511 H5 MOL 392 −5.023 5.147 −0.384 H 1512 H16 MOL 392 −6.737 6.227 0.099 H 1513 H17 MOL 392 −8.35 6.264 −0.625 H 1514 H18 MOL 392 −8.144 5.672 1.036 H 1515 H13 MOL 392 −9.222 3.536 0.478 H 1516 H14 MOL 392 −9.53 4.37 −1.067 H 1517 H15 MOL 392 −8.761 2.774 −1.072 H

Example 3—Structure-Based Design of Potent Tau Assembly Inhibitors

Overview

The molecular simulations performed in Example 2 suggest that LMT is able to bind dGAE and enhance the stabilisation of a compact folded conformation. The inventors hypothesized that molecules which are able to bind tightly within this pocket would further stabilise dGAE and prevent assembly into PHF. They therefore set out to analyse the pharmacophore features of the LMT binding pocket.

Methods

A pharmacophore model was designed by analysing the interactions between LMT and the LMT binding pocket identified in Example 2. A series of compounds were designed to test the binding hypothesis generated from this model. These compounds were tested in a cell-based tau aggregation assay as described in Rickard J E, Horsley D, Wischik C M, Harrington C R., Methods Mol Biol. 2017; 1523:129-140, using LMT (EC₅₀=0.096 μM) as a reference compound.

Results

As shown on FIG. 12D, the LMT binding pocket is predominately hydrophobic in nature, with some sites suitable for forming hydrogen bonds. The site is capped by phenylalanine residues Phe378 and Phe346, and contains other hydrophobic side chains in Val350, Leu315, Ile354, Ile371. A number of side chains have the potential of forming hydrogen-bonds to a molecule within the pocket. These include the backbone of Lys347, the hydroxy group of Thr373, the backbone carbonyl of Leu315 and the NH of Glu372. LMT forms interactions with Lys347 and Thr373. LMT is also bound by Lys343, which has the potential to form Pi-cation interactions with the aromatic rings of LMT (see LMT structure, FIG. 12C).

Using in silico design approaches, a set of compounds were designed that exploit the hydrogen-bonding features observed with LMT, and the shape and lipophilic features of the LMT-induced binding pocket. These compounds were assessed in a cell-based Tau aggregation assay. The features of these compounds and the results of the cell-based Tau aggregation assays are shown in Table 2. All compounds had the same central ring and one of 6 substituents to the left of the central ring and one of 5 substituents to the right of the central ring (by comparison to the central ring, left and right rings or LMT shown on FIG. 12C). Compounds 15-18, 12-14, 9-11. 3-8 and 1-2, respectively, had the same substituent on the right. Compounds (3, 9, 15), (4, 10), (1, 5, 12, 16), (6, 13), (7, 14, 17) and (2, 8, 11, 18) respectively, had the same substituent on the left. The substituents were designed to test the required strength of hydrogen bonds with the pocket, as well as optimal orientation for hydrophobic and pi interactions. The results confirm the key features of the predicted stabilised cryptic pocket that can be exploited to inhibit Tau aggregation. Compound 17 showed the best activity with an EC₅₀=0.139 μM. This compound fulfilled a number of binding features, including hydrogen-bonding to the Glu372 backbone NH, hydrogen-bonding to the Lys374 backbone NH and a hydrogen bond to the NH of Thr373. Additionally, the compound comprises an aromatic substituent that binds in the lipophilic pocket towards Phe378, forming a face-edge Pi stack with Phe378, and it is well positioned to interact with the Lys343 through Pi-cation interactions.

The results suggest that interactions with Lys343 is important to differentiate compound potency, with both hydrogen bonding to the backbone carbonyl of Lys343 and Pi-cation interactions with Lys343 are favourable for ligand potency. Small substituents that are able to bind in the lipophilic pocket towards Phe378, and the ability to form a H-bond with the NH of Thr373 also appear relevant. The structural differences between compounds 17 and 18 include the removal of the aromatic substituent that binds in the lipophilic pocket towards Phe378, and the introduction of a substituent on one of the heavy atoms that formed a H bond with the NH of Thr373. These changes bring about a marked 16-fold loss in potency, compound 18 (EC₅₀=2.246 μM). The structural differences between compounds 17 and 14 include a reorientation of the right substituent (the left substituent in these compounds being identical), which affect the hydrogen-bonding orientation and hence strength between the left substituent of the ligand and the protein. The interaction with Phe378 is also lost. These changes result in a loss of potency by 8-fold (EC₅₀=1.17 μM for compound 14).

The structure-activity relationships of the 19 compounds examined (including LMT) supports the hypothesis that a stabilised cryptic pocket exists which can be exploited to achieve tau assembly inhibition.

TABLE 2 Compounds tested for tau aggregation inhibition Activity Compound Predicted interactions (average distance between heavy atoms) (EC50, μM) LMT H-donor with THR373 sidechain OG1 (average distance 0.096 between NH and OG1 of THR373 = 2.92) H-acceptor with LYS347 backbone NH (average distance between S and NH of LYS347 = 3.45) pi-H with interaction SER352 backbone CA (average distance between 6-ring and CA of SER352 = 4.1) pi-cation interaction LYS343 sidechain NZ (average distance between aromatic ring and NZ of LYS343 = 3.5) 17 H-donor with GLU372 sidechain OE1 (average distance 0.139 between H donor atom and OE1 of GLU372 = 3.52) H-donor with LEU315 backbone C═O (average distance between H donor atom and C═O of LEU315 = 3.17) H-acceptor with ILE371 backbone CA (average distance between H acceptor atom and CA of ILE371 = 3.25) H-acceptor with SER341 sidechain CB (average distance between H acceptor atom and CB of SER341 = 3.28) H-acceptor with LYS343 backbone NH (average distance between H acceptor atom and NH of LYS343 = 2.86) H-acceptor with PHE346 backbone CA (average distance between H acceptor atom and CA of PHE346 = 3.4) H-acceptor with LYS347 backbone NH (average distance between H acceptor atom and NH of LYS347 = 2.99) pi-H with LYS343 sidechain CB (average distance between 5- ring and CB of LYS343 = 4.21) pi-cation with LYS343 sidechain NZ (average distance between aromatic ring and NZ of LYS343 = 3.67) 12 H-donor with GLU372 sidechain OE1 (average distance 0.204 between H donor atom and OE1 of GLU372 = 3.07) H-acceptor with ILE371 backbone CA (average distance between H acceptor atom and CA of ILE371 = 3.24) H-acceptor with GLU372 backbone NH (average distance between H acceptor atom and NH of GLU372 = 2.98) H-acceptor with LYS343 backbone NH (average distance between H acceptor atom and NH of LYS343 = 3.42) H-acceptor with LYS347 backbone NH (average distance between H acceptor atom and NH of LYS347 = 3.02) 16 H-acceptor with LYS347 backbone NH (average distance 0.239 between H acceptor atom and NH of LYS347 = 3.14) Hydrophobic interactions with the sidechains of Leu315, Val350, Ile371 1 H-donor with GLU372 sidechain OE1 (average distance 0.381 between H donor atom and OE1 of GLU372 = 2.97) H-acceptor with ILE371 backbone CA (average distance between H acceptor atom and CA of ILE371 = 3.32) H-acceptor with GLU372 sidechain NH (average distance between H acceptor atom and NH of GLU372 = 3) H-acceptor with GLU342 backbone NH (average distance between H acceptor atom and NH of GLU342 = 3.17) H-acceptor with PHE346 backbone CA (average distance between H acceptor atom and CA of PHE346 = 3.45) H-acceptor with LYS347 backbone NH (average distance between H acceptor atom and NH of LYS347 = 3.11) 7 H-donor with LEU315 backbone C═O (average distance 0.463 between H donor atom and C═O of LEU315 = 3.22) H-acceptor with ILE371 backbone CA (average distance between H acceptor atom and CA of ILE371 = 3.26) H-acceptor with GLU372 backbone NH (average distance between H acceptor atom and NH of GLU372 = 2.94) H-acceptor with GLU342 backbone NH (average distance between H acceptor atom and NH of GLU342 = 3.16) H-acceptor with PHE346 backbone CA (average distance between H acceptor atom and CA of PHE346 = 3.4) H-acceptor with LYS347 backbone NH (average distance between H acceptor atom and NH of LYS347 = 3.05) 3 H-donor with LYS343 backbone C═O (average distance 0.583 between H donor atom and C═O of LYS343 = 3.25) H-donor with THR373 sidechain OG1 (average distance between H donor atom and OG1 of THR373 = 3.12) 4 H-donor with THR373 sidechain OG1 (average distance 0.591 between H donor atom and OG1 of THR373 = 3.34) H-acceptor with LYS369 backbone CE (average distance between H acceptor atom and CE of LYS369 = 2.98) H-acceptor with GLU372 backbone NH (average distance between H acceptor atom and NH of GLU372 = 3.11) 9 H-donor with THR373 sidechain OG1 (average distance 0.682 between H donor atom and OG1 of THR373 = 3.04) Hydrophobic interactions with Val350 and Leu315 and Ile371 5 H-acceptor with ILE371 backbone CA (average distance 0.757 between H acceptor atom and CA of ILE371 = 3.28) H-acceptor with GLU372 backbone NH (average distance between H acceptor atom and NH of GLU372 = 2.98) H-acceptor with GLU342 backbone NH (average distance between H acceptor atom and NH of GLU342 = 3.14) H-acceptor with LYS343 backbone NH (average distance between H acceptor atom and NH of LYS343 = 3.47) H-acceptor with PHE346 backbone CA (average distance between H acceptor atom and CA of PHE346 = 3.4) H-acceptor with LYS347 backbone NH (average distance between H acceptor atom and NH of LYS347 = 3.09) 11 pi-H with LYS343 sidechain CB (average distance between 5- 0.926 ring and CB of LYS343 = 3.84) Hydrophobic interactions with Val350 and Leu315 and Ile371 14 H-donor with GLU372 sidechain OE1 (average distance 1.17 between H donor atom and OE1 of GLU372 = 2.87) H-donor with LEU315 backbone C═O (average distance between H donor atom and C═O of LEU315 = 3.13) H-acceptor with LYS347 backbone NH (average distance between H acceptor atom and NH of LYS347 = 3.14) pi-H with LYS343 sidechain CB (average distance between 5- ring and CB of LYS343 = 3.66) pi-cation with LYS343 sidechain NZ (average distance between aromatic ring and NZ of LYS343 = 3.63) 2 H-donor with GLU372 sidechain OE1 (average distance 1.397 between H donor atom and OE1 of GLU372 = 3.05) H-acceptor with LYS343 backbone NH (average distance between H acceptor atom and NH of LYS343 = 3.1) H-acceptor with LYS347 backbone NH (average distance between H acceptor atom and NH of LYS347 = 2.97) pi-H with LYS343 sidechain CB (average distance between 5- ring and CB of LYS343 = 4.04) 8 H-acceptor with ILE371 backbone CA (average distance 1.684 between H acceptor atom and CA of ILE371 = 3.27) H-acceptor with GLU372 backbone NH (average distance between H acceptor atom and NH of GLU372 = 2.96) H-acceptor with GLU342 backbone NH (average distance between H acceptor atom and NH of GLU342 = 3.14) H-acceptor with PHE346 backbone CA (average distance between H acceptor atom and CA of PHE346 = 3.35) H-acceptor with LYS347 backbone NH (average distance between H acceptor atom and NH of LYS347 = 3.09) 10 H-donor with THR373 sidechain OG1 (average distance 1.732 between H donor atom and OG1 of THR373 = 3.21) H-acceptor with LYS369 backbone NZ (average distance between H acceptor atom and NZ of LYS369 = 3.39) pi-H with LYS343 backbone NH (average distance between aromatic ring and NH of LYS343 = 4.44) pi-H with LYS343 sidechain CB (average distance between 5- ring and CB of LYS343 = 4.04) 6 H-donor with LEU315 backbone C═O (average distance 1.896 between H donor atom and C═O of LEU315 = 3.38) H-acceptor with ILE371 backbone CA (average distance between H acceptor atom and CA of ILE371 = 3.33) H-acceptor with LYS343 backbone NH (average distance between H acceptor atom and NH of LYS343 = 3.15) pi-H with LYS343 sidechain CE (average distance between 6- ring and CE of LYS343 = 3.7) 13 H-acceptor with ILE371 backbone CA (average distance 1.903 between H acceptor atom and CA of ILE371 = 3.26) H-acceptor with GLU372 backbone NH (average distance between H acceptor atom and NH of GLU372 = 2.96) H-acceptor with LYS343 backbone NH (average distance between H acceptor atom and NH of LYS343 = 3.24) H-acceptor with PHE346 backbone CA (average distance between H acceptor atom and CA of PHE346 = 3.3) H-acceptor with LYS347 backbone NH (average distance between H acceptor atom and NH of LYS347 = 3.01) 15 H-donor with GLU372 sidechain OE1 (average distance 1.981 between H donor atom and OE1 of GLU372 = 3.32) H-donor with THR373 sidechain OG1 (average distance between H donor atom and OG1 of THR373 = 3.01) 18 H-donor with LYS343 backbone C═O (average distance 2.246 between H donor atom and C═O of LYS343 = 3.57) H-acceptor with PHE346 backbone CA (average distance between H acceptor atom and CA of PHE346 = 3.46) H-acceptor with LYS347 backbone NH (average distance between H acceptor atom and NH of LYS347 = 3.21)

Example 4—Mechanism for dGAE Aggregation into Paired Helical Filaments

Overview

Molecular dynamics simulation was used to study the assembly of dGAE in a conformation that is able to bind LMT, into a paired helical filament (PHF) arrangement.

Methods

The parameterised 3D protein structures of the PHF 10 oligomer (stack of 10 monomers) modelled from PDB ID: 5O3L (as explained in Example 1), was used as a reference template to model the approach of a LMT-free dGAE monomer (as determined using Tau97 in Example 2) towards the PHF and then complete assembly. First the system was truncated from 97 to 73 residues according to the sequence reported in the literature (Fitzpatrick et al., 2017), which describes based on cryo-EM investigation of tau filaments that filament cores are made of two identical protofilaments comprising residues ³⁰⁶V-to-F³⁷⁸ of the Tau protein. This truncated system will be referred to as dGAE73 and PHF73 for convenience. The LMT-free dGAE73 monomer was obtained from the LMT MD simulation experiment (Example 2) and truncated appropriately. The C-terminal truncated amino acids were left as free acids and the N-terminal residues as free bases. Following an established protocol for MD simulations, system minimisation followed by an equilibration run, in total 1,200 ns, and a production MD simulation was then performed for 258 ns. A structure was extracted from the simulation at 50 ns which represented an average of the flexibility observed in the system over the 258 ns, see FIG. 13 .

The 50 ns timeframe structure was allowed to assemble following the simulated annealing approach to nudged elastic band (NEB) method using the AMBER software. The 50 ns timeframe was used as a starting reference structure and the fully assembled and minimised state was used as a final reference structure. A total of 144 replica frames were employed in constructing the trajectory for assembly.

The NEB simulation was run in a number of stages, 1) Heating up the system, 2) simulated annealing and equilibration, and finally 3) slow cooling. These are described below.

During the heating stage, the system was allowed to warm up from 0 to 300K, performed with a small spring constant. A run of 20 ps of MD was performed with a 0.5 fs time step (nstlim=40000, dt=0.0005). The SHAKE algorithm was not used (ntc=1, ntf=1). Since the coordinates of the end points are fixed in Cartesian space in NEB and the intervening structures cannot move far due to the springs there is no need to re-centre the coordinates every few hundred steps so this option was turned off (NSCM=0). The generalised Born model was used for the implicit solvent with a sodium chloride salt concentration of 0.2M (igb=1, saltcon=0.2). Nonbonded cutoffs and truncated Born radii calculations were obtained by setting these to 999.0 angstroms (cut=999.0, rgbmax=999.0). The NEB specific options used were (ineb=1 [turn on neb], skmin=10, skmax=10 [spring constants]). The simulation was started with a relatively small spring constant of 10 KCal/mol/Å². For temperature regulation the langevin thermostat (ntt=3) was used together with a high collision rate of 1000 ps-1 (gamma_ln=1000). NMR weight restraints (nmropt=1) were used to linearly increase the value of temp0 from 0.0 to 300.0 K over 35,000 steps of the 40,000 step simulation (istep1=0, istep2=35000, value1=0.0, value2=300.0). This means that at step 0 the value of the target temperature (temp0) will be 0.0 K. At step 5,000 it will be 42.86 K, at 10,000 it will be 85.72 K, by 20,000 it will be 171.44 K and by 35,000 it will be at 300.0 K. It will then remain at 300.0 K until the end of the 40,000 step run.

In the simulated annealing and equilibration stage, a run of 100 ps of MD at 300K with a larger spring constant was used. The coordinates were saved at a frequency of once every 5,000 steps (ntwx=5000). The next step is to run a simulated annealing run. The NMR restraints option was used to control the value of temp0 during the run. A total of 300 ps of MD was run with the following temperature profile:

-   -   0-50 ps: Heat from 300 K to 400 K     -   50-100 ps: Equilibrate at 400 K     -   100-150 ps: Heat from 400 to 500 K     -   150-200 ps: Equilibrate at 500 K     -   200-250 ps: Cool to 300 K     -   250-300 ps: Equilibrate at 300 K

The final stage is to slowly cool the system down and this was performed in two stages. In the first stage the system was gradually cooled down in 50 K steps over 120 ps using the following profile:

-   -   0 to 10 ps:300K->250 K     -   10 to 20 ps: Equil at 250 K     -   20 to 30 ps: 250 K->200 K     -   30 to 40 ps: Equil at 200 K     -   40 to 50 ps: 200->150 K     -   50 to 60 ps: Equil at 150 K     -   60 to 70 ps: 150 K->100 K     -   70 to 80 ps: Equil at 100 K     -   80 to 90 ps: 100 K->50 K     -   90 to 100 ps: Equil at 50 K     -   100 to 110 ps:50 K->0 K     -   110 to 120 ps: Equil at 0 K

Finally, to calculate a final energy minimization of the path the velocity Verlet algorithm was used. This was performed in a one final 200 ps long stage of cooling where setting temp0=0.0 K completes the final quenched MD and having turned on the quenched velocity Verlet method (vv=1).

Results

In an effort to further evaluate the hypothetical LMT-stabilised conformation of dGAE, we used molecular dynamics simulations to study the assembly of PHFs from a conformer of dGAE which is able to bind LMT. A PHF arrangement of 5 stacked dGAE73 (assembled into a 10 monomers PHF, referred to herein as PHF73) oligomers and a monomer dGAE73 in the stabilised state were modelled in a fully solvated environment, as shown on FIG. 2 , and in FIGS. 13 and 14A. The simulation was performed in two steps, as described in the Methods above. The first step was to search for the initial site of anchoring the monomer dGAE73 onto the PHF using traditional molecular dynamics simulations in explicit solvent. The second step was to examine the PHF formation using nudged elastic band (NEB) molecular dynamics simulations.

In the first stage (anchor stage), 13 molecular dynamics simulations were started and the orientation of alignment of the dGAE73 monomer relative to the PHF73 stack was observed. The starting orientation was randomly chosen, and the monomer was placed beyond hydrogen-bonding distance to the PHF73 stack. The dGAE73 monomer aligned itself over the residues Val337-Gly355 which form a tight hairpin (as shown on FIGS. 14A-14B) in a number of the simulations. This sequence of 19 residues contains numerous charged amino acids residues, including 4 acidic groups, 5 basic groups and 3 polar side chains. This result may be the result of long-range electrostatic forces pulling the monomer towards to the PHF stack. Once the hairpin (residues 337-355) is in contact with the PHF stack the hairpin (residues 337-355) tightly anchors itself. Over a simulation period of over 40 ns freely mobile N- and C-terminal arms of the dGAE73 monomer unravel from the tightly bound stable core, as is illustrated in FIGS. 13 and 14D.

Then, after a period of 50 ns of production molecular dynamics simulation, a nudged elastic band (NEB) molecular dynamics simulation was used to find the minimum energy path for the rearrangement into the fully assembled PHF73 conformation. The observed folding pathway is shown on FIG. 15 .

A key step in the formation of PHF73 is the formation of alternating positively charged and negatively charged sidechain stacks in the hairpin loop of PHF73 (residues Val337-Gln355), as best seen on FIG. 16B. This anchors the dGAE73 monomer to the PHF73. This is followed by another key step, the flipping of Pro332 from a trans to a cis configuration, at around frame 80, then back to a cis configuration, at around frame 130, as illustrated on FIG. 17 . This enables the formation of interactions between His329, His330 and Lys331 of the monomer and the stack, zipping together from the C- to the N-terminal over a short period (see FIGS. 15 D-E). The N- and C-termini of the dGAE73 monomer then continue to unravel and the PHF73 formation completes in a zipper-like fashion, starting from Ile360-Thr361 (see FIGS. 15E-F). This occurs in two directions, first from 355-360 in the C- to N-direction, then from 361-367 in the N- to C-direction. Lastly, the cross-beta sheet conformation is formed, starting from Phe378 to Asn368 and closing the residues from C- to N-terminal, and joining residues 318-306 of the N-terminal in the C- to N-direction.

Through this process, the N-terminal arm (residues Val306-Lys321), closely coupled to the C-terminal arm (residues Gly367-Phe378), of dGAE73 collapses onto the PHF73 driven by electrostatic interactions between dGAE73 and the PHF73. Indeed, temporary hydrogen bonds between Asp314 in dGAE73 and Lys370 in the PHF73 stack and between Gln307 of dGAE73 and Lys375 and Thr377 of the PHF753 stack assist the zipper-like closure, helping to bring together the terminal arms of the dGAE73 and PHF73. During the zipper closure, the preferential binding conformation is maintained through hydrophobic interactions between the two arms (C- and N-ter) of the monomer. Indeed, the hydrophobic residues Ile308, Tyr310 and Pro312 of the N-terminal dGAE73 create well-packed hydrophobic interactions with the C-terminal residues of dGAE73 including Leu375 and Phe378, as shown on FIG. 18 . The key residues mentioned above are highlighted in the sequence below (SEQ ID NO: 4 (dGAE73, human/mouse, 73 amino acids), together with indices showing the order of the steps mentioned above: [V₃₀₆QVYKPVDLSKV₃₁₈]⁶TSKCGSLGNI[H₃₂₉H₃₃₀K₃₃₁]³[P₃₃₂]²GGGQ[V₃₃₇EVKSEKLDFKDRVQ SKIG₃₅₅]¹ [SLDNI₃₆₀]⁴ [T₃₆₁HVPGGG₃₆₇]5[NKKIETHKLTF₃₇a]⁶

where the indices refer to:

-   -   Step 1: anchoring of monomer to PHF by formation of alternating         positively charged and negatively charged sidechain stacks in         the hairpin loop of PHF (residues Val337-Gln355).     -   Step 2: flipping of Pro332.     -   Step 3: formation of interactions between His329, His330 and         Lys331 of the monomer and the stack, residues 319-331 zip         together in a direction from C- to the N-terminal.     -   Step 4: zipping from 355-360 in the C- to N-direction.     -   Step 5: zipping from 361-367 in the N- to C-direction.     -   Step 6: formation of cross-beta sheet conformation, starting         from Phe378 and closing the residues from C- to N-terminal, and         joining residues 318-306 of the N-terminal in the C- to         N-direction.

A crystal structure of a hexapeptide corresponding to residues V₃₀₆QIVYK₃₁₁ underlined in the above sequence was obtained by Sawaya M R, et al. (2007). This structure was used to design peptide inhibitors of VQIVYK aggregation by Sievers S A, et al. (2011) and Zheng J, et al. (2011). The PHF6 (306VQIVYK311) peptide has been shown to be sufficient for in vitro polymerization to filamentous structures and microcrystals (Goux et al., 2004; Sawaya et al., 2007; von Bergen et al., 2000 and 2001). The PHF6 motif is located in the repeat regions of the microtubule binding domain of tau and has been suggested to play a prominent role in the formation of PHFs and is also part of the PHF core composed of cross-β structure (47-48). Our observations support the finding that the PHF6 sequence in dGAE does form a cross-β structure, although it occurs at the end of the assembly stage and in our experiments is seen to be the last step in completing the stabilisation of the PHF, the closing of the zip.

More recently, the structure of another hexapeptide that is not part of dGAE (VQIINK) was determined and used to design inhibitors of aggregation based on the finding that VQIINK forms a more extensive steric zipper interface than VQIVYK. These inhibitors, peptides WINK and MINK, were found to reduce Tau40 aggregation.

Based on the modelling above, the peptide inhibitors of VQIVYK aggregation would at most be able to prevent the formation of the very last step of the aggregation process.

Example 5—Examination of the Folding Pathway Using mAb Experiments

Overview

In this example, the inventors identified the core region of dGAE filaments (using protease digestion experiments) and monitored the progression of self-assembly of dGAE into filaments by following the surface exposure of specific epitopes to a range of monoclonal antibodies (using immuno-precipitation and ELISA experiments) were performed to examine the folding pathway and the effect that LMT had on the process.

Methods

Protein production: Recombinant tau297-391 (dGAE) was produced and purified from ‘E. coli as previously described in phosphate buffer (20 mM, pH 7.4) (AI-Hilaly et al, 2017) and stored at −20° C. until required.

Filament assembly: dGAE (100 μM) was incubated at 37° C. in phosphate buffer (20 mM, pH7.4), with or without DTT (10 mM) and, with or without agitation (700 rpm), for 24 hours.

Methylthioninium chloride (MTC): Methylthioninium chloride (MTC) was provided by TauRx Therapeutics Ltd. The concentrations are expressed as free methylthioninium base (MT), and MTC added as the specified time with or without DTT.

Protease digestion: 100 μM dGAE with 10 mM DTT in phosphate buffer (20 mM, pH 7.4), with or without MTC at ratios up to 1:5 was agitated at 700 rpm, 37° C. for 72 hours. 10 μg aliquots were digested with increasing concentrations of Proteinase K (PK), Pronase E (PE), or left untreated. The digestion was carried out at 37° C. for 1 hour then the reaction was stopped with 1 mM Pefabloc and samples were placed on ice for 5 minutes. Sample buffer without any reducing agent was added, incubated for 5 minutes at room temperature then loaded onto a gel.

Immunogold labelling transmission electron microscopy: For all dilutions of antibodies and secondary gold probes a modified phosphate buffer saline (PBS, pH 8.2) was used; this buffer was supplemented with BSA (1%), Tween-20 (0.005%), 10 mM Na EDTA, and NaN3 (0.2 g/I). dGAE-C322A (100 μM) was incubated with and without MT (10 μM) and incubated for 24 h at 37 C with agitation at 700 oscillations per min in dark. Preformed fibrils were decorated ‘on grid’ using a polyclonal anti-tau antibody (Sigma-Aldrich, SAB4501831). In summary, 4 μl of dGAE-C322 fibrils were pipetted onto Formvar/carbon coated 400-mesh copper TEM support grids (Agar Scientific, Essex, UK) and left for 1 min, then a filter paper was used to remove the excess. Normal goat serum (1.10 in PBS, pH 8.2) was used for blocking for 15 min at room temperature. Grids were then incubated with (10 μg/ml IgG) rabbit anti-tau polyclonal antibody for 2 h at room temperature, rinsed three times for two minutes using PBS (pH 8.2), and then immunolabeled in a 10-nm gold particle-conjugated goat anti-mouse IgG secondary probe (GaR10 British BioCell International, Cardiff, UK; 1.10 dilution) for 1 h at room temperature. The grids were then rinsed five times for two minutes in PBS (pH 8.2) and rinsed five times for two minutes in distilled water. Finally, the grids were negatively stained using 0.5% uranyl acetate. As a negative control, Aß342 fibrils were examined using the same protocol.

SDS-PAGE: SDS-PAGE was conducted on the entire assembly mixtures as well as the supernatant and pellet fractions used for CD (3 μl of each per lane). Samples were mixed with SDS-PAGE sample buffer (without reducing agent) and separated using Any kDa Mini-Protean™ TGX™ Precast gels (Bio-Rad) at 120 V, until the sample buffer reached the end of the gel. The gel was stained using Imperial Protein Stain (Thermo Scientific), following the manufacturer's instructions, before sealing the gel and scanning on a Canon ImageRunner Advance 6055i scanner.

Immunoblotting: A stock solution of recombinant dGAE was diluted to 100 μM in phosphate buffer (20 mM, pH7.4) with 10 mM DTT. The solution was agitated at 700 rpm, 37° C. and aliquots taken at t0 (following a 10 second vortex), 2, 4, 6, 8, 24 and 72 hours and stored at −20° C. When all the time points had been collected, samples were vortexed well and 3 μL of the whole mixture applied to a nitrocellulose membrane, 1.5 μL at a time, allow to almost dry then the next 1.5 μL applied. The membrane was washed for 2 minutes in TBS-T (tris buffered saline with 0.05% tween), 2 minutes in TBS then blocked in 5% milk in TBS-T for 1 hr. scAbs were diluted in block and incubated with the membrane for 1 hour followed by 3×10 minute washes in TBS-T. Secondary antibody (anti-Human GHRP, Invitrogen) diluted 1:1000 in block was then added to the membrane for 1 hour then washed 3×10 min TBS-T. ECL reagent (BioRad) was applied to the membrane for 3 minutes, drained then imaged using a scanner with a maximum exposure time of 10 minutes.

Results

dGAE Filaments Contain a Protease Resistant Core.

Soluble or fibrils of dGAE (100 μM) in reducing conditions (10 mM DTT) were incubated with increasing concentrations of proteinase K or Pronase E then samples were run on SDS-PAGE. Soluble dGAE runs as a doublet at 10/12Kda under reducing conditions and no bands of this size were observed in the presence of either PK or PE. Soluble dGAE in the supernatant was completely digested with even the lowest concentration of enzyme tested (25 μg/mL). The pellet contains mostly fibrillar dGAE and runs as a monomer ad dimer on SDS-PAGE. With PK, a protease resistant band at around 8 kDa was observed with enzyme concentrations up to around 100 μg/mL, after which the band disappeared indicating the core was fully digested. For PE the core withstood even up to 500 μg/mL enzyme. Mass spectrometry analysis of both protease resistant bands (see FIGS. 22A-B) revealed a protected core region of H299-K370 with a theoretical molecular weight of 7.5 kDa (data not shown).

Repeating these experiments in the presence of LMT revealed that LMT prevents the formation of a protease resistant core. Proteolysis revealed that soluble dGAE is digested while filaments retain a protease resistant core. In the absence of DTT, dGAE remains able to self-assemble and remains protease resistant (data not shown) while in the presence of DTT, LMT is able to prevent inhibition and the resulting soluble dGAE can be digested (FIG. 22C).

Assembly of dGAE Results in Progressive Loss of Epitopes

A series of monoclonal single chain antibodies (scAbs) raised to recognise different regions of the peptide were used to perform epitope mapping to follow the conformational change that accompanies self-assembly. Immunoblots were performed on 100 uM dGAe with 10 mM DTT dGAE assembled in reducing conditions over 72 hours at different time points. These were repeated 10 times and the data was pooled to produce graphical output normalised to the intensity at time 0 and error bars were provided to show the variation in the data (see FIG. 19 ). In particular, dGAE (100 μM, 20 mM phosphate buffer pH7.4 with 10 mM DTT) was agitated at 37° C. for 72 hours in the absence or presence of LMT (reduced) (1:5 ratio). Samples were withdrawn at 2, 4, 6, 8, 10, 20, 40 and 60 hours of agitation and 3 μL placed on a nitrocellulose membrane. Membrane was rinsed with TBS-T and incubated with antibodies binding to residues 319-331 (AB1), 337-355 (AB2), 355-367 (AB3), 367-378 (AB4), 379-391 (AB5), 379-390 (AB6), and 306-359 (AB7) for 1 hour followed by HRP-conjugated anti-sheep antibody for 1 hour and washed again washed with TBS-T x5. Detection was performed using Clarity ECL blotting substrate (BioRad) and exposed to X-ray film. The results of these experiments are shown on FIG. 19 , for the experiments without LMT. The epitopes bound by the antibodies AB1-AB6 are highlighted in the sequence below (residues 3 to 97 of SEQ ID NO: 4; also referred to herein as dGAE, SEQ ID NO: 3, human/mouse, 95 amino acids)—with indices referencing the antibody corresponding to the epitope in bold between brackets. The sequence below also shows the sequence of dGAE underlined.

I₂₉₇HVPGGGSVQIVYKPVDLSKV[T₃₁₉SKCGSLGNIHHK₃₃₁]^(AB1)PGGGQ[V₃₃₇EVKSEKLDFKDRV QSKI{G₃₅₅]^(AB2)SLDNITHVPGG[G₃₆₇}^(AB3)NKKIETHKLTF₃₇₈ ]^(AB4)[{R₃₇₉ENAKAKTDHGA₃₉₀}^(AB6)E₃₉₁]^(AB5)

Binding Profiles in the Absence of LMT

As best seen on FIG. 19B, at time 0 (before agitation), the antibodies AB2 (337-355), AB4 (367-378) and AB5 (379-391) are able to bind dGAE, indicating that their epitopes are exposed. The epitope of AB3 (355-367) appears to be less exposed. In order to elucidate this, the results of the NEB simulation described in Example 4 were used to compute the distance between the conformation of the epitopes AB1 to AB4 as they progress through the aggregation process, and the conformation of these epitopes at the start or the end of the aggregation process. The results of this analysis are shown on FIGS. 20B-C. This analysis revealed that in the stable free dGAE conformation (starting point of the simulations in Example 4), the epitope of AB3 (355-367) is partially occluded by residues 321-343. Further, this analysis revealed that the epitope of AB3 initially moves further away from the final bound conformation in the first 10 frames of the simulation, then “jumps” to a conformation close to that found in the fully assembled PHF around frame 133. This is in marked contrast with the behaviour of the epitopes of AB2 (337-355) and AB4 (367-378), which do not show this initial behaviour in the first 10 frames of the simulation. The conformations close to that in the fully assembled PHF are expected to enable slightly more binding of AB3 (355-367), which could explain the increase in signal in the dot blot experiments from 20 to 70 hours (see FIG. 19C).

Turning now to AB1 (319-331), the signal from the Dot Blot experiments implies that this epitope conformation is less exposed at t0 than AB2-5 (see FIG. 19B). This antibody recognises 323-328, which is found at the PHF-dimer interface and shows low affinity for the soluble protein at time 0 h and this is lost by 2 h. As shown on FIG. 20B, the conformation of this epitope changes rapidly (in the first 10 frames) during assembly, which is reflected in a rapid loss of signal in the Dot Blot experiments.

Turning to AB2 (337-355), in the context of PHF structure (Fitzpatrick, 2017) is found at the b-helix (see FIG. 19A). As the protein folds into the PHF structure, recognition of this region reduces gradually but the Dot Blot data indicates that this epitope may be in a conformation suitable for binding of the antibody throughout assembly (see FIG. 19B-C). The simulation data is in accordance with this, since the conformation of this epitope is within 3 Å of the final fully bound conformation for a significant part of the assembly (see frames 50 onwards on FIG. 20B).

The short AB4 (367-378, which binds within the C-shape region of the PHF, see FIG. 19A) epitope sequence appears to be bind to the antibody effectively throughout PHF assembly (see FIG. 19B-C, where the signal only gets as low as 0.5). Analysis of the RMSD of the AB4 sequence in dGAE during the assembly does not show a marked change in RMSD, the maximum RMSD is about 4 Å with an average close to 2 Å (see FIG. 20B). Further, the AB4 sequence is close to the C-terminal flexible jaws of the PHF. This inherent flexibility may allow detection by the antibody, albeit not optimally, throughout the PHF assembly. This flexibility of the C-terminal is reflected in the similar responses in the epitopes AB7 (306-359) and AB5 (379-390), see FIG. 19 .

Together with the data from Example 4, this data confirms that the epitope of AB2 (337-355) is first to bind to PHF, from the middle of the sequence at R349. This is followed by the binding of charged groups E342, D348, followed by the sequences of V350-Q351-K343, Q336-V337-K347. The PGGG repeat between the epitopes of AB1 (319-331) and AB2 (337-355) then binds, followed by residues 1360-T261 in the epitope of AB3 (355-367). Then slowly the C-terminal end of AB1 (319-331) binds sequentially towards to N-terminal end, and the epitope of AB4 (367-378) then binds to PHF. This starts with starts with K375 and then amino acids on both sides are assembled. The final sequences to bind is the P364-GG-G367 sequence in the epitope of AB3 (355-378).

From these experiments, we can infer that as dGAE follows a sequence whereby the association of two molecules initiates the assembly (AB1, 323-328) followed by a folding into a C-shape (AB3, 358-364) and the formation of the b-helical hairpin. The structure then curves to bury (AB4, 370-378) and (AB6, 379-391) regions at the centre and C-termini.

Immunogold labelling of soluble and preformed filaments of dGAE confirmed that the AB3 recognition sequence is exposed in soluble dGAE and buried in PHF (see FIG. 23A). In fact, none of the antibodies are able to access dGAE PHF (see FIG. 23B). Furthermore, AB3 does not recognise PHF in AD human brain tissue.

LMT Mediated Inhibition of dGAE Aggregates and Restoration of Immunoreactivity

The truncated core repeat domain dGAE (297-391), is the predominant fragment that constitutes bulk of the PHF core in AD (Wischik et al, 1988). During dGAE aggregation in vitro, scAb binding regions on dGAE are ‘hidden’ or ‘occluded’ which leads to a loss of immunoreactivity in aggregation samples. Here we have shown the occlusion of binding regions in aggregated dGAE samples and the recovery of immunoreactivity in the presence of LMTM, a tau-aggregation inhibitor. The scAbs tested for binding are core region specific with binding regions given in Table 3 below). For preparing the aggregates, 10 μl 10 mM DTT was added to 1000 μl 100 μM dGAE and agitated with/without LMTM (1:5 ratio) at 700 rpm for 24 h at 37° C. Following overnight agitation, one third of each sample was kept aside as ‘total’ and the rest was spun down at 16000×g for 30 min and separated into ‘supernatant’ and ‘pellet’. The pellet was then resuspended in half the original volume for further experiments. The immunoreactivities of core region specific scAbs towards dGAE aggregates formed with/without LMTM was tested using a sandwich ELISA format using a ‘E’ specific monoclonal antibody 423 mAb. This mAb has been shown to specifically bind to the Pronase resistant core structure in the PHFs (Wischik et al, 1988). ELISA plates were coated with 10 μg/ml 423 mAb and blocked as normal. Doubling dilutions of dGAE aggregate ‘total’, ‘supernatant’ and ‘pellet’ samples at 10 μg/ml starting concentration were added to designated wells in doubling dilutions in 1×PBS. dGAE monomer (non-aggregated) was included as assay control. All double dilutions were done in final volumes of 100 μl. This was left to incubate on lab bench for 1 h followed by the addition of test scAbs at 10 μg/ml. Anti-HuCk HRP labelled secondary antibody was added as described previously and ELISA data generated is represented using the graph below.

TABLE 3 Core binding scAbs tested in epitope occlusion assays and their specific binding regions on Ht40 Binding regions scAbs tested on hT40 AB2 337-355 AB3 355-367 AB5 360-390 AB1 319-331 AB7 297-356 AB8 367-379

All scAbs tested showed increased binding to aggregated dGAE ‘total’ and ‘supernatant’ samples, when aggregation was conducted in the presence of LMTM. This proves the opening or revealing of occluded antibody binding regions on dGAE where LMTM is preventing the aggregation event, leading to an increased immunoreactivity (FIG. 21 ).

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All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and or to the other particular value. Similarly when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. 

1. A method for selecting or designing a compound for modulating the aggregation of a Tau protein or a truncated form thereof, the method comprising using computer-implemented molecular modelling means to: compare the three-dimensional structure of a candidate compound with a three-dimensional structure of at least a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or a variant or derivative thereof that is structurally equivalent to said part; and determine whether the candidate compound is able to simultaneously form non-covalent interactions with two or more of Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350, Ser352, Ile354, Lys369, Ile371, Glu372, Phe378 and Thr373, of SEQ ID NO:1, or equivalent amino acids in a variant or derivative, wherein a candidate compound that is able to form said interactions is predicted to modulate the aggregation of the Tau protein or truncated form thereof.
 2. The method of claim 1, comprising determining whether the candidate compound is able to simultaneously form non-covalent molecular interactions with Lys343 and Glu372 of SEQ ID NO:1.
 3. The method of claim 1, wherein the compound is for inhibiting the aggregation of a Tau protein or a truncated form thereof into paired helical filaments, and wherein the compound is optionally a small molecule, a peptide, a polypeptide or a combination thereof.
 4. (canceled)
 5. The method of claim 1, wherein the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 comprises (i) a binding pocket that is capped by Phe378 and Phe346, exposes the hydrophobic side chains of residues Val350, Leu315, Ile354 and/or Ile371, and contains residues Leu315, Lys343, Lys347, Glu372, and/or Thr373 capable of forming hydrogen bonds to a molecule within the binding pocket; (ii) a hairpin loop between residues Val337 and Gly355 of SEQ ID NO:1; (iii) the sequence Pro364-Gly367 located between a loop formed by the sequence Tyr219-Lys331 and the sequence Pro332-Gly335; (iv) is stabilized at least in part by hydrogen bonds between Glu342 and Val318 and/or Thr319, between Gly367 and Lys340, between Asn368 and Lys340, between Lys369 and Glu372, between Lys370 and Asp358, between Ile371 and Ser316, between Glu372 and Ser356, between Thr373 and Gln351, Ser316, between His374 and Gln351, and between Lys375 and Gln351; and/or (v) is that represented by the structure co-ordinates in Table 1, or a structure modelled on these coordinates. 6-9. (canceled)
 10. The method of claim 1, wherein the three-dimensional structure of the part of the Tau protein is obtainable by performing molecular dynamics simulations of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 in the presence of LMT to obtain one or more complex conformations and selecting a three-dimensional structure of the part of the Tau protein by applying a stability criterion and a binding affinity criterion to the one or more complex conformations; optionally wherein the stability criterion applies to the distance between complex conformations in consecutive frames of the molecular dynamics simulation after a predetermined amount of time, and/or wherein the binding affinity criterion applies to the value of a placement scoring function <−80 kcal/mol or a scoring function <−8.5 as determined by the GB IV scoring function within the CCG MOE docking software.
 11. (canceled)
 12. The method of claim 1, further comprising designing a pharmacophore model, wherein the pharmacophore includes features representative of non-covalent molecular interactions with two or more of: Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350, Ser352, Ile354, Lys369, Ile371, Glu372, Phe378 and Thr373, of SEQ ID NO:1.
 13. The method of claim 1, wherein the part of the Tau protein: (i) comprises amino acids 306-378 of SEQ ID NO:1; (ii) comprises amino acids 297-391 of SEQ ID NO:1; (ii) comprises amino acids 295-391 of SEQ ID NO:1; (iv) consists of amino acids 297-391 of SEQ ID NO:1; (v) consists of amino acids 295-391 of SEQ ID NO:1; or (vi) consists of amino acids 306-378 of SEQ ID NO:1.
 14. (canceled)
 15. The method of claim 1, further comprising repeating the steps of comparing and determining with a further candidate compound that differs from the previous candidate compound in at least one substituent.
 16. The method of claim 1, wherein comparing the three-dimensional structure of a candidate compound with a three-dimensional structure of at least a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or a variant or derivative thereof that is structurally equivalent to said region comprises computing the interaction energy between the candidate compound and the part of the Tau protein represented in the three-dimensional structure.
 17. A computing system comprising a processor and a memory storing machine-readable instructions that, when executed by the processor, cause the processor to implement the method of claim
 1. 18. A computer-implemented method for evaluating the ability of a candidate compound to bind to a binding pocket of a Tau protein, the method comprising the steps of: (a) receiving the three-dimensional structure coordinates of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or equivalent amino acids in a variant or derivative thereof that is structurally equivalent to said part, wherein the three-dimensional structure of amino acids 315-378 of SEQ ID NO: 1 comprises the binding pocket of the Tau protein; (b) performing a fitting operation between a candidate compound and the binding pocket; and (c) analysing the results of the fitting operation to determine whether the candidate compound is able to bind to the binding pocket.
 19. The method of claim 18, wherein the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 is that represented by the structure co-ordinates shown in Table 1, or a structure modelled on these coordinates.
 20. The method of claim 18, wherein the candidate compound is able to bind to the pocket if it is able to simultaneously form non-covalent molecular interactions with two or more of Leu315, Ser341, Glu342, Lys343, Phe346, Lys347, Val350, Ser352, Ile354, Lys369, Ile371, Glu372, Phe378 and Thr373, of SEQ ID NO:1; optionally with Lys343 and Glu372 of SEQ ID NO:1.
 21. (canceled)
 22. The method of claim 18, wherein the binding pocket is capped by Phe378 and Phe346, exposes the hydrophobic side chains of residues Val350, Leu315, Ile354 and/or Ile371, and contains residues Leu315, Lys343, Lys347, Glu372, and/or Thr373 capable of forming hydrogen bonds to a molecule within the binding pocket.
 23. The method of claim 18, wherein the three-dimensional structure of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 (i) comprises a hairpin loop between residues Val337 and Gly355 of SEQ ID NO:1; (ii) comprises the sequence Pro364-Gly367 located between a loop formed by the sequence Tyr219-Lys331 and the sequence Pro332-Gly335; or (iii) is stabilised at least in part by hydrogen bonds between Glu342 and Val318, Thr319, between Gly367 and Lys340, between Asn368 and Lys340, between Lys369 and Glu372, between Lys370 and Asp358, between Ile371 and Ser316, between Glu372 and Ser356, between Thr373 and Gln351, Ser316, between His374 and Gln351, between Lys375 and Gln351. 24-25. (canceled)
 26. The method of claim 18, wherein the three-dimensional structure coordinates of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or equivalent amino acids in a variant or derivative thereof that is structurally equivalent to said part have been obtained by performing molecular dynamics simulations of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 in the presence of LMT to obtain one or more complex conformations and selecting a complex conformation using a stability criterion and a binding affinity criterion.
 27. The method of claim 18, wherein step (a) receiving the three-dimensional structure coordinates of a part of the Tau protein comprises obtaining the three-dimensional structure coordinates of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or equivalent amino acids in a variant or derivative thereof that is structurally equivalent to said part by: (a) performing molecular dynamics simulations of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 in the presence of LMT to obtain one or more complex conformations that differ in their three-dimensional conformations; and (b) selecting a complex conformation using a stability criterion and a binding affinity criterion, wherein the three-dimensional structure coordinates of a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 are defined as the three-dimensional structure coordinates of the part of the Tau protein in the selected complex conformation; optionally wherein the stability criterion applies to the distance between conformations in consecutive frames of the molecular dynamics simulation after a predetermined amount of time, and/or wherein the binding affinity criterion applies to the value of a docking score.
 28. The method of claim 18, wherein the part of the Tau protein comprises amino acids 306-378 of SEQ ID NO:1, wherein the part of the Tau protein comprises amino acids 297-391 of SEQ ID NO:1, wherein the part of the Tau protein comprises amino acids 295-391 of SEQ ID NO:1, wherein the part of the Tau protein consists of amino acids 297-391 of SEQ ID NO:1, wherein the part of the Tau protein consists of amino acids 295-391 of SEQ ID NO:1, or wherein the part of the Tau protein consists of amino acids 306-378 of SEQ ID NO:1.
 29. The method of claim 18, wherein the three-dimensional structure coordinates of the part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or equivalent amino acids in a variant or derivative thereof that is structurally equivalent to said part correspond to a conformation that has the following structural characteristics: hydrogen bonds between Glu342 and Val318 and/or Thr319; optionally wherein the hydrogen bonds are between the Glu342 carboxylic acid and the backbone NH of Val318 and the sidechain OH of Thr319; one or more hydrogen bonds between one or more of residues Lys369-Thr377 and one or more of residue Ser341-Gln351; optionally wherein the one or more bonds comprise: (i) a bond between Gln351 and Thr373, preferably wherein the bond is between the backbone carbonyl of Gln351 and the hydroxyl sidechain of Thr373; (ii) a bond between Gln351 and His374, preferably wherein the bond is between the sidechain carbonyl of Gln351 and the sidechain amine of His374; (iii) a bond between Gln351 and Lys375, preferably wherein the bond is between the sidechain carbonyl of Gln351 and the backbone amine of Lys375; (iv) a bond between Arg349 and Thr377, preferably wherein the bond is between a sidechain amine of Arg349 and the hydroxyl sidechain of Thr377, between a sidechain amine of Arg349 and the hydroxyl backbone of Thr377, and/or between the carbonyl backbone of Arg349 and the hydroxyl sidechain of Thr377; (v) a bond between Glu372 and Ser356, preferably wherein the bond is between the carboxylic acid side chain of Glu372 and the backbone NH of Ser356, or between the carboxylic acid side chain of Glu372 and the OH-sidechain of Ser356; and/or (vi) a bond between Glu372 and Lys369, preferably wherein the bond is between the carboxylic acid side chain of Glu372 and the NH sidechain of Lys369; no beta sheets; a hairpin loop comprising residues Val337-Gly355; the PGGG sequence formed by residues Pro364-Gly367 is within a distance of 13 A of the PGGG sequence Pro332-Gly335 and/or within a distance of 2 A of a loop formed by the sequence Thr319-Lys331; the PGGG sequence formed by residues Pro364-Gly367 is located between the PGGG sequence Pro332-Gly335 and a loop formed by the sequence Thr319-Lys331; residues Lys369-Thr377 are within a distance of 6 Å of residues Asp314-Ser316, optionally wherein the distance between the Ser316 beta-carbon and Thr373 backbone carbonyl is between 2.5 Å and 5.0 Å; residues Gly355-Gly367 and Asn368-Arg379 are within 2 A hydrogen bonding distance; Glu338 is folded towards Val363, optionally wherein the distance (RMSD) between the carbonyl oxygen side chain of Glu338 and the backbone amine nitrogen of Val363 NH during the final 10 ns of a 50 ns simulation is between 2 Å and 4 Å, or below 5 Å; the total water accessible surface area calculated for the part of the protein is at least 20% lower than the corresponding values calculated for a conformation as provided by the three-dimensional coordinates with PDB identifier 5O3L; and/or the polar and/or hydrophobic accessible surface area(s) calculated for the part of the protein is/are at least 20% lower than the corresponding values calculated for a conformation as provided by the three-dimensional coordinates with PDB identifier 5O3L.
 30. A method for selecting or designing a compound for modulating the aggregation of a Tau protein or a truncated form thereof, the method comprising: using a three-dimensional structural model of at least a part of the Tau protein comprising amino acids 315-378 of SEQ ID NO:1 or a variant or derivative thereof that is structurally equivalent to said part, wherein the model is an intermediate in the aggregation process of the part of the Tau protein with a paired helical filament (PHF), wherein the model is generated by simulating the conformational changes of the part of the Tau protein from a compact folded state to a an aggregated state such that: (i) residues Val337-Gln355 form a hairpin loop that moves to align with alternating positively charged and negatively charged sidechain stacks in the hairpin loop of the PHF; (ii) residue Pro332 switches between a trans and a cis configuration; (iii) residues 355-378 and 306-318 move to form stabilising cross-β sheets with corresponding residues of the PHF through hydrophobic zippering; and generating a model of a complex of the compound and the intermediate.
 31. The method of claim 30, wherein the compact folded state has (i) any of the structural characteristics defined in claim 29 and/or (ii) the structure co-ordinates shown in Table 1, or a structure modelled on these coordinates.
 32. (canceled)
 33. The method of claim 30, wherein generating a model of a complex of the compound and the intermediate comprises identifying the compound as binding to the intermediate and preventing the occurrence of any of steps (i) to (v), optionally any of steps (i) to (iii).
 34. (canceled) 